Hormone-induced rise in cytosolic Ca2 in axolotl hepatocytes: properties of the Ca2 influx channel
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
Hormone-induced rise in cytosolic Ca21 in axolotl
hepatocytes: properties of the Ca21 influx channel
THOMAS LENZ AND JOCHEN W. KLEINEKE
Abteilung Klinische Biochemie, Zentrum Innere Medizin,
University of Göttingen, 37075 Göttingen, Germany
Lenz, Thomas, and Jochen W. Kleineke. Hormone- Very recently, investigations on Drosophila melano-
induced rise in cytosolic Ca21 in axolotl hepatocytes: proper- gaster have drawn attention to certain proteins (trp,
ties of the Ca21 influx channel. Am. J. Physiol. 273 (Cell trpl) with an apparent capacity of both channel forming
Physiol. 42): C1526–C1532, 1997.—Calcium entry in nonex- and the sensing of the filling state of the endoplasmic
citable cells occurs through Ca21-selective channels activated reticulum Ca21 store (32, 33). Hence, the Drosophila
secondarily to store depletion and/or through receptor- or
second messenger-operated channels. In amphibian liver,
store-operated channel has been put forward as a
hormones that stimulate the production of adenosine 38,58- model for capacitative Ca21 entry. Analogous proteins,
cyclic monophosphate (cAMP) also regulate the opening of an however, have not been detected in liver (43).
ion gate in the plasma membrane, which allows a noncapaci- In variance, in fish and amphibian liver, the effect of
tative inflow of Ca21. To characterize this Ca21 channel, we adrenergic agonists and vasotocin is mediated via the
studied the effects of inhibitors of voltage-dependent Ca21 generation of cAMP (19, 20, 42), and not via InsP3. Yet,
channels and of nonselective cation channels on 8-bromoa- in parenchymal liver cells from axolotl (Ambystoma
denosine 38,58-cyclic monophosphate (8-BrcAMP)-dependent mexicanum), hormones that stimulated cAMP forma-
Ca21 entry in single axolotl hepatocytes. Ca21 entry provoked tion (the order of efficacy was glucagon . isoprenaline .
by 8-BrcAMP in the presence of physiological Ca21 followed epinephrine $ arginine vasotocin) also provoked a
first-order kinetics (apparent Michaelis constant 5 43 µM at pronounced increase in cytosolic Ca21, which was not
the cell surface). Maximal values of cytosolic Ca21 (increment
due to a mobilization of the cation from internal stores
,300%) were reached within 15 s, and the effect was tran-
sient (half time of 56 s). We report a strong inhibition of by InsP3/thapsigargin, but to an increased inflow from
cAMP-dependent Ca21 entry by nifedipine [half-maximal the extracellular medium. Thus, in axolotl liver, in
inhibitory concentration (IC50 ) 5 0.8 µM], by verapamil contrast to rat liver, hormones that stimulate the
(IC50 5 22 µM), and by SK&F-96365 (IC50 5 1.8 µM). production of cAMP also regulate the opening of an ion
Depolarizing concentrations of K1 were without effect. Gado- gate in the plasma membrane, which allows an inflow
linium and the anti-inflammatory compound niflumate, both of Ca21 (and Mn21 ). The effect is rather specific, since
inhibitors of nonselective cation channels, suppressed Ca21 guanosine 38,58-cyclic monophosphate (cGMP) failed to
influx. This ‘‘profile’’ indicates a novel mechanism of Ca21 induce Ca21 entry (23). We have proposed that this
entry in nonexcitable cells. channel could belong to the category of second messen-
adenosine 38,58-cyclic monophosphate; second messenger- ger-operated Ca21 channels, as defined by Meldolesi
operated calcium channel; calcium channel pharmacology; and Pozzan (29). In nonexcitable tissues, such channels
SK&F-96365; fenamates have so far only been found in blood cells (10, 27, 36).
The aim of this investigation was to further character-
ize the nature of this cAMP-activated Ca21 channel of
axolotl liver cells, using a variety of compounds that
THE REGULATION OF GLYCOGEN breakdown in mamma- influence Ca21 entry in excitable and nonexcitable cells:
lian liver by a-adrenergic agonists and vasoactive the phenylalkylamine verapamil, the dihydropyridine
peptides has been extensively studied. These hormones nifedipine, both potent inhibitors of Ca21 entry in heart
generate as second messengers diacylglycerol and inosi- and skeletal muscle, and the imidazole derivative
tol 1,4,5-trisphosphate (InsP3 ); the latter mobilizes SK&F-96365, which inhibited receptor-mediated Ca21
Ca21 from the endoplasmic reticulum and in addition entry (as compared with receptor-mediated Ca21 re-
triggers Ca21 entry into the cell (5). In most cells, lease) in nonexcitable cells (human platelets, neutro-
including hepatocytes (18, 22, 26), the rate of Ca21 phils, and endothelial cells) and which has been used as
influx after hormonal stimulation seems to be con- a tool to discriminate between voltage-gated Ca21 entry
trolled by the filling state of internal InsP3-sensitive and receptor-mediated Ca21 entry in GH3 and artery
Ca21 stores (34). When such stores are depleted, an smooth muscle cells (30). Because lanthanides (1027 to
inflow of Ca21 is triggered by a mechanism that may 1025 M) block stretch- and receptor-activated nonselec-
depend on the presence of Ca21, InsP3, and/or inositol tive cation channels, but also Ca21 entry through
1,3,4,5-tetrakisphosphate, or other yet to be defined voltage-dependent channels (15), we investigated the
diffusible factors (reviewed in Ref. 6). In rat liver, the effect of Gd31 on cAMP-dependent Ca21 entry. As an
rate of Ca21 entry into cells via store-operated channels additional inhibitor of nonselectivc cation channels in
may be enhanced if glucagon or other adenosine 38,58- membranes, we examined the effect of niflumate, a
cyclic monophosphate (cAMP)-generating hormones are nonsteroidal anti-inflammatory drug (12, 15).
present during a challenge with Ca21-dependent hor- Using single-cell dual-wavelength epifluorescence
mones (7, 28). The nature of this mechanism is obscure measurements of cytosolic Ca21 in amphibian hepato-
at the present. cytes, we report a strong inhibition of cAMP-dependent
C1526 0363-6143/97 $5.00 Copyright r 1997 the American Physiological SocietyINHIBITION OF CA21 INFLOW BY DIHYDROPYRIDINES C1527
Ca21 entry by SK&F-96365 [half-maximal inhibition A (plus additions as specified) in a petri dish (Falcon 3001)
concentration (IC50 ) 5 1.8 3 1026 M], by the dihydropyri- with a central quartz window (diameter 5 15 mm). Water-
dine nifedipine (IC50 5 8 3 1027 M), and by verapamil. insoluble compounds were prepared as concentrated stock
Furthermore, the lanthanide Gd31 and niflumate, both solutions in dimethyl sulfoxide (DMSO). The concentration of
DMSO in the petri dish never exceeded 1% (vol/vol). The same
potent inhibitors of nonselective cation channels, sup-
amount of DMSO was added to control incubations.
pressed Ca21 influx. It is concluded that in axolotl Ca21 measurements were performed on single hepatocytes
hepatocytes the rise in intracellular Ca21 after hor- using a fura 2 data aquisition system (Luigs and Neumann,
monal stimulation is due to a Ca21 inflow via a novel Ratingen, Germany) mounted to an inverted microscope
dihydropyridine- and SK&F-96365-sensitive nonselec- (Zeiss IM 35) equipped with epifluorescence, a xenon lamp
tive cation channel. (Osram, XBO 75 W/2), a rotating filter wheel (357/380- to
390-nm excitation, 480- to 540-nm emission), and a photomul-
MATERIALS AND METHODS tiplier (Hamamatsu 928 SF). The sampling rate was 2/s. For a
more detailed description and evaluation of the equipment,
Materials. Fura 2 acetoxymethyl ester (AM) was purchased
see Neher (31). Calibration of the system was done using
from Molecular Probes (Eugene, OR). SK&F-96365 51-(b-[3-(4-
fluorescent beads.
methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidaz-
Application of agonists. Application of agonist [8-bromoad-
ole hydrochloride6 was a kind gift from SmithKline Beecham
enosine 38,58-cyclic monophosphate (8-BrcAMP)] was done
Pharmaceuticals (Welwyn, UK). BAY K 8644 was from Bayer.
using a microcapillary to direct a flow of solution of agonist
Collagenase (‘‘Worthington’’ type CLS II, 206 U/mg) came
under constant pressure (1,000 hPa) from a distance of ,30
from Biochrom (Berlin, Germany). 3-Aminobenzoic acid ethyl
µm for 5–10 s onto the equatorial surface of the single cell
ester (MS-222) was from Sigma (Munich, Germany). All other
under investigation. The capillary (2–3 µm diameter) was
chemicals were of analytical grade and were obtained from
positioned using an Eppendorf ECET 5170 micromanipula-
Merck (Darmstadt, Germany).
tor, and an ECET microinjection system (Eppendorf 5242)
Isolation of hepatocytes. Axolotls (A. mexicanum) were
coupled to the capillary was activated for the time and
maintained in aerated water tanks at 20°C. The animals were
pressure specified to generate the flow of agonist. All other
fed twice weekly on fish pellets (Fisch-Fit, Interquell Stärke,
compounds were dissolved in medium A and were present in
Wehringen, Germany) and had a body weight of 60–80 g
the ‘‘bath’’ (petri dish) at concentrations given in Figs. 1–5.
when used. Results from both males and females are pre-
The rate of Ca21 increase (nM/s) and the maximum level in
sented together, because there were no differences observed
cytosolic Ca21 (DCa21 ) were calculated from fura 2 recordings
between sexes (19).
of individual hepatocytes. The initial rate of Ca21 increase
The animals were anesthetized by immersion in 0.05%
reflects the rate of Ca21 entry into single hepatocytes and is
(wt /vol) MS-222. The cannulation and extirpation of the liver
related to the proportion of active (‘‘open’’) channels in the
were as described previously (19, 23).
membrane. The initial rate is largely independent of signal
Hepatocytes were prepared using Ca21-free amphibian
distortion by compensating mechanisms (desensitization). At
Krebs-Ringer bicarbonate buffer (aKRB) (80 mM NaCl, 3 mM
high concentrations of inhibitor(s), the determination of this
KCl, 0.6 mM KH2PO4, 0.8 mM MgSO4, and 16 mM NaHCO3,
value was more reliable.
pH 7.4) as the perfusate. Briefly, the liver was perfused via
If not otherwise stated, values given are means 6 SE from
the portal vein for 15 min with the above medium in an open
7–10 single cells exposed and stimulated individually under
perfusion, and then after readdition of CaCl2 (1 mM) and
identical conditions per dish. The experiments were repeated
collagenase (0.05 g/100 ml), the perfusion was continued for
at least three times with independent cell preparations.
40–50 min in a recirculating system. After this step, the liver
was minced, and the disintegrating tissue fragments as well RESULTS
as separated single cells were collected and passed through a
double layer of cheesecloth. This suspension was washed Kinetics of cAMP-dependent Ca21 uptake. 8-BrcAMP
three times with aKRB by centrifugation (100 g for 1 min). (1 mM) when applied from the outside using a microin-
Usually .85% of the cells were viable as judged by trypan jection glass capillary for 5 s from a distance of ,30 µm
blue exclusion (0.2% trypan blue, 1% bovine serum albumin onto the surface of single axolotl hepatocyte led after a
in aKRB). short delay to an increased influx of Ca21, as shown for
Measurement of cytosolic Ca21. The cells were washed once six of seven individual hepatocytes in the same petri
(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
dish (Fig. 1). In most cells, the increase of cytosolic Ca21
D-glucose, and 20 mM N-2-hydroxyethylpiperazine-N8-2-
was transient with a half-life of decay of ,1 min (57 6 4
ethanesulfonic acid, pH 7.4 (medium A) to give a concentra- s, n 5 6). Some cells however exhibited longer-lasting
tion of ,60 mg wet wt /ml. They were incubated with fura responses, some also with superimposed oscillations
2-AM (5 µM) for 30 min at 25°C in a shaking water bath (100 (not shown). Maximum levels of cytosolic Ca21 (DCa21:
cycles/min). After this, the cells were spun down (1 min, 100 211 6 20 nM; n 5 5) were obtained within 15 s (rate:
g), the supernatant was discarded, and the pellet was resus- 13 6 1.5 nM/s; n 5 6).
pended in the same volume of medium A and further incu- The rate of Ca21 influx and the maximum increase of
bated for up to 30 min at 4°C. Thereafter, the cells were cytosolic Ca21 of cells treated as defined above were
washed twice with medium A (100 g, 1 min) and resuspended dependent on the concentration of 8-BrcAMP (Fig. 2,
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
A and B). The influx of Ca21 followed first-order kinet-
allow deesterification of fura 2-AM. The latter was controlled ics with an apparent affinity constant of 8.6 3 1024 M.
during this period by monitoring the epifluorescence of single Maximal levels of cytosolic Ca21 were observed at a
hepatocytes at 360-nm excitation. Ca21 concentration was 8-BrcAMP concentration of 1–2 mM. These data com-
calculated from the fluorescence ratio 360/380 nm (31). pare favorably with earlier measurements of hepato-
Hepatocytes (1–2 mg wet wt) were suspended in 2 ml medium cytes in suspension (23). Because of the experimentalC1528 INHIBITION OF CA21 INFLOW BY DIHYDROPYRIDINES
the availability of extracellular Ca21, as has been
reported earlier (23).
Effect of SK&F-96365 on cAMP-dependent Ca21 en-
try. The imidazole derivative SK&F-96365 has been
introduced as a tool to discriminate between voltage-
gated Ca21 entry and receptor-mediated Ca21 entry
(30). SK&F-96365 inhibited cAMP-dependent Ca21 in-
flow in axolotl hepatocytes in a dose-dependent man-
ner. The dose-response curves for the rate of Ca21 entry
and for the maximal increase are shown in Fig. 3. The
IC50 values for SK&F-96365 were 1.4 and 1.7 3 1026 M
for the rate of Ca21 entry and maximal increase (DCa21 ),
respectively.
Effect of dihydropyridines and verapamil on cAMP-
dependent Ca21 entry. The dihydropyridine (8, 9) nifedi-
Fig. 1. Stimulation of Ca21 entry in presence of 8-bromoadenosine
pine, when tested under comparable conditions, inhib-
38,58-cyclic monophosphate (8-BrcAMP) in single hepatocytes. Iso- ited markedly the cAMP-dependent Ca21 influx (IC50 5
lated hepatocytes from axolotl were loaded with fura 2 acetoxymethyl 8 3 1027 M). This sensitivity is 20–50 times more
ester as given in MATERIALS AND METHODS. Hepatocytes equivalent to pronounced than that reported for liver by others (18,
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, 26). BAY K 8644, an agonistic dihydropyridine, which
10 mM D-glucose, and 20 mM N-2-hydroxyethylpiperazine-N8-2- binds during the open state of L-type Ca21 channel and
ethanesulfonic acid, pH 7.4 (medium A). Cells were allowed to settle prolong their open time (24), when present in equimolar
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
concentration had no additional effect (Fig. 4A, open
of a single hepatocyte (marked in graph by small vertical lines). square). BAY K 8644 at 2.5 µM on its own, however,
Epifluorescence recorded at 360 and 390 nm and corresponding Ca21 increased the basal Ca21 by 17% and cAMP (1 mM)-
trace of 7 individual cells exposed and stimulated in succession are dependent DCa21 by 47%.
depicted.
The potency of the phenylalkylamine verapamil to
block Ca21 entry was about one order of magnitude
topology (distance between cell and mound of the lower (IC50 5 2.2 3 1025 M) than that of nifedipine.
capillary, pressure, and time), the effective concentra-
Dihydropyridines are the ‘‘classical’’ inhibitors of
tion of agonists is ,20-fold more diluted at the cell
L-type Ca21 channels that are abundant in electrically
surface compared with the concentration in the capil-
lary. Hence, an apparent minimal effective concentra- excitable tissues, like muscle and brain cells. These
tion of 25 µM (Fig. 2A, inset, intersection with the cells are depolarized in the presence of K1. In experi-
abscissa) is equivalent to a concentration of 1–2 3 1026 ments in which KCl was applied at a concentration of
M at the cell surface. The resting Ca21 concentration of 100 mM onto single axolotl hepatocytes, we could not
axolotl hepatocytes was 85 nM (Fig. 2B, inset, intersec- detect any effect of such depolarizing concentrations of
tion with the ordinate). At saturating concentrations of KCl on intracellular Ca21 (data not shown).
agonist, a rise in cytosolic Ca21 by ,300% is observed. Effect of Gd31 on cAMP-dependent Ca21 entry. The
8-BrcAMP at a concentration of 1 mM in the micropi- lanthanide Gd31 inhibited cAMP-dependent Ca21 entry
pette (,50 µM at the cell surface) was nearly maxi- very efficiently. A 50% inhibition of the rate of Ca21
mally effective in most cell preparations under these entry and of the maximal increase of cytosolic Ca21 was
conditions. This effect of 8-BrcAMP was dependent on observed at a concentrations of 2.5 3 1026 M (Fig. 5).
Fig. 2. Effect of 8-BrcAMP on rate of
Ca21 entry and cytosolic Ca21 concen-
tration in single hepatocytes. Condi-
tions were as given in Fig. 1 except that
8-BrcAMP was applied at concentra-
tions given on abscissa. Rate of Ca21
increase (A) and maximal increment
(B) in cytosolic Ca21 (DCa21 ) were calcu-
lated from fluorescence recorded. Note:
inset in B shows initial changes in
cytosolic Ca21. All other conditions were
as given in MATERIALS AND METHODS.INHIBITION OF CA21 INFLOW BY DIHYDROPYRIDINES C1529
Fig. 3. Inhibition of 8-BrcAMP-depen-
dent Ca21 entry by SK&F-96365. Condi-
tions 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 microcapil-
lary. Rate of Ca21 increase (A) and
maximal increment (B) in cytosolic Ca21
(DCa21 ) were calculated from fluores-
cence recorded. For better comparison,
data from 6 independent cell prepara-
tions were normalized. Values for 100%
were as follows: A, 14 6 3 nM/s (n 5 6);
B, 180 6 19 nM (n 5 6).
Effect of niflumate on cAMP-dependent Ca21 entry. has been observed (30). The mechanism of this inhibi-
Niflumate inhibited cAMP-dependent Ca21 entry by tion is still elusive. The proposal, however, that cyto-
,90% (rate: 9.6 and 7.2%; DCa21: 13.6 and 13.2% of chrome P-450 may link intracellular Ca21 stores with
control at 1 or 5 3 1024 M niflumate, respectively). plasma membrane influx (2) has been questioned by
others (36). In axolotl liver, we could ex-
DISCUSSION
clude a participation of intracellular, capacitative stores
Using dual-wavelength excitation epifluorescence in cAMP-dependent Ca21 influx, which is in support of
measurements of Ca21 on single hepatocytes, we demon- a cytochrome P-450-independent interaction (23).
strate here unique properties of this Ca21-conducting Opposing effects of SK&F-96365 on HL-60 cells have
channel in axolotl hepatocytes (a nonexcitable splanch- been recently reported by Leung et al. (25). At low
nic cell). concentrations (,16 µM), SK&F-96365 inhibited Ca21
The entry of Ca21 evoked by cAMP was strongly entry, whereas at higher concentrations (16–100 µM),
inhibited by the imidazole derivative SK&F-96365 it provoked release of intracellular Ca21, and by this
(IC50 ,2 3 1026 M), whereas that after microinjection of promoted even Ca21 entry (30–100 µM). The latter
InsP3 was inhibited only at concentrations .1024 M could be inhibited by La31, but not by nifedipine.
(data not shown). This inhibition is about one order of The comparably sensitive inhibition of cAMP-depen-
magnitude more effective than that described for a dent Ca21 entry observed in the presence of the dihy-
variety of different cells including rat hepatocytes dropyridine derivative nifedipine was not expected.
(0.8–3 3 1025 M, see Refs. 10, 11, 25, 30, 40). SK&F- Dihydropyridines are known to block rather selectively
96365, which belongs to a group of imidazole antimy- L-type voltage-dependent Ca21 channels of excitable
cotics that have been originally used to block cyto- tissues (9), a channel type which is absent in hepato-
chrome P-450 but also Ca21 and Ca21-dependent K1 cytes, as judged by electrophysiological measurements
channels (3), was introduced as a novel inhibitor of (37) or Northern analysis (17). This is confirmed by our
receptor-mediated Ca21 entry into cells (30). In addi- failure to demonstrate Ca21 entry after membrane de-
tion, inhibition of voltage-gated Ca21 entry in GH3 and polarization in the presence of K1 (100 mM), which
rabbit ear artery smooth muscle cells by SK&F-96365 reveals that the channel decribed here although sensi-
Fig. 4. Inhibition of 8-BrcAMP-dependent
Ca21 entry by nifedipine and verapamil. Nifed-
ipine (r) or verapamil (l) was present in bath
at concentrations indicated (abscissa). BAY K
8644 plus nifedipine was present in equimolar
concentration (k). 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 6 2 nM/s (n 5 6); B, 170 6 18 nM (n 5 6).C1530 INHIBITION OF CA21 INFLOW BY DIHYDROPYRIDINES
order of magnitude used to block voltage-dependent
L-type Ca21 channels of excitable cells in vitro, i.e.,
1–10 µM.
The inhibition of cAMP-dependent Ca21 entry ob-
served in axolotl hepatocytes in the presence of niflu-
mate or the lanthanide Gd31 was not surprising. Both
compounds are potent inhibitors of Ca21 entry through
nonselective cation channels (Ref. 15 and references
therein). Because of an ionic radius close to that of Na1
and Ca21, Gd31 (0.2–100 µM) can block efficiently
stretch- or receptor-activated nonselective cation chan-
nels (4, 11, 40) but, like La31, also voltage-dependent
channels (39).
The nonsteroidal anti-inflammatory fenamates have
Fig. 5. Inhibition of 8-BrcAMP-dependent Ca21 entry by Gd31. been applied to block Ca21 entry via nonselective cation
Conditions were as given in legend to Fig. 3, except that GdCl3 was channels in cells from rat exocrine pancreas (12), in
present in bath at concentrations indicated. Values for 100% were human polynuclear leukocytes (21), and in mucosa-
13 6 2 nM/s (n 5 2) (solid bars) and 176 6 19 nM (n 5 2) (open bars)
for rate and increment, respectively.
type mast cells (35). Apart from nonselective cation
channels, Cl2 channels are blocked by fenamates (13,
35). In mucosa-type mast cells, the Cl2 channel blocker
tive to dihydropyridines lacks certain properties of a 4,48-diisothiocyanostilbene-2,28-disulfonic acid, how-
classical L-type channel of excitable cells, in particu- ever, fully obstructed Cl2 currents without affecting
lar, the ability of voltage sensing, a property which Ca21 influx, thus indicating that the effect of niflumate
is thought to be located on the S4 segment of the on Ca21 influx may be dissociated from that on Cl2
a1-subunit (9). channels (35).
Ca21 influx channels of nonexcitable cells sharing As discussed above, all compounds used here, ex-
these properties have been recently found in B lympho- cept for nifedipine and verapamil, are to a variable
cytes from rat, which showed dihydropyridine but no degree inhibitory on capacitative Ca21 entry (11, 16, 21,
voltage sensitivity (1), and in an erythroleukemia cell 25, 26, 40).
line from mouse, where a truncated a1-subunit lacking The agonist-induced Ca21 influx in axolotl hepato-
the first four transmembrane segments was expressed cytes may be characterized as follows. The influx
(27). Furthermore, the trp/trpl gene product from Dro- depends totally on the generation of cAMP, which in
sophila that forms a nonselective cation channel pre- turn acts indirectly via protein phosphorylation cata-
sumably involved in capacitative Ca21 entry in inverte- lyzed by protein kinase A (23). The influx of Ca21
brates and vertebrates shows some sequence homology measured in the presence of 8-BrcAMP as a surrogate
to the voltage-operated Ca21 channel a1-subunit, but follows first-order kinetics, with a maximal rate of ,60
lacks arginine residues of the S4 region (33, 41). nM/s and an apparent Michaelis constant of ,5 3 1026
The phenylalkylamine verapamil inhibited Ca21 M 8-BrcAMP, as calculated for the concentration pre-
entry in axolotl hepatocytes (50% effective concentra- sent on the cell surface. Protein phosphorylation(s)
tion 5 22 µM) but in comparison with nifedipine with could be coupled to and/or modulate the open state of an
lower sensitivity. In contrast to dihydropyridines, phe- ion-gating channel in the membrane, as demonstrated
nylalkylamines enter the cell to interact with a high- for voltage-gated ion channels (8, 9). The pharmacologi-
affinity binding protein on the endoplasmic reticulum, cal profile of the Ca21 influx channel in amphibian
which has been identified in guinea pig and human hepatocytes reveals certain relationships to these chan-
liver (14). As for nifedipine, the effects of verapamil nels, as well as to nonselective cation channels. The
reported so far for liver (and hepatocytes) are rather Ca21 entry shows a remarkable dihydropyridine sensi-
controversial. Studying capacitative Ca21 entry, Llopis tivity but lacks the ability of voltage sensing, indicating
et al. (26) failed to see effects of verapamil or nifedipine certain homologies to the dihydropyridine binding site
(up to 50 µM), whereas Striggow and Bohnensack (38) of the a1-subunit, but apparently differences in the S4
observed an incomplete inhibition of this Ca21 entry segment. Examples of other nonexcitable cells sharing
mechanism at verapamil or diltiazem concentrations these properties have been discussed above. This rela-
between 200 and 400 µM. Others have reported com- tionship is reinforced by the distinct effects of verapa-
plete inhibition of 45Ca21 exchange across the liver cell mil and SK&F-96365 or Gd31, because the selectivity of
plasma membrane in the presence of 50–100 µM the latter compounds for voltage-gated Ca21 entry and
nifedipine or verapamil (18). A stretch-activated nonse- nonselective cation channels appears to be low (30, 39).
lective cation channel found in rat hepatocytes and rat The sensitivity to SK&F-96365, Gd31, and niflumate,
hepatoma cells was not affected by nifedipine, verapa- all of which act by different mechanisms (15), discloses
mil, or La31 (4). the properties of a receptor-activated nonselective cat-
The effect of nifedipine (or verapamil) shown here on ion channel.
axolotl hepatocytes appears to be more specific, since This novel dihydropyridine-sensitive channel, which
the effective concentration of nifedipine (1–5 µM) is the to our knowledge is absent in rodent liver, could serveINHIBITION OF CA21 INFLOW BY DIHYDROPYRIDINES C1531
as an example for a diversity of types and subtypes of 19. Janssens, P. A., J. Kleineke, and A. G. Caine. Calcium-
channels in various tissues and species. independent stimulation of glycogenolysis by arginine vasotocin
and catecholamines in liver of the axolotl, Ambystoma mexica-
The generous gift of axolotls by Prof. Dr. W. Hanke, Karlsruhe, num. J. Endocrinol. 109: 75–84, 1986.
Germany, is gratefully acknowledged. 20. Janssens, P. A., and P. Lowrey. Hormonal regulation of hepatic
Address for reprint requests: J. W. Kleineke, Abt. Klin. Biochemie glycogenolysis in the carp, Cyprinus carpio. Am. J. Physiol. 252
Zentrum Innere Medizin, Univ. of Göttingen, Robert-Koch-Str. 40, (Regulatory Integrative Comp. Physiol. 21): R653–R660, 1987.
37075 Göttingen, Germany. 21. Kankaanranta, H., and E. Moilanen. Flufenamic and tolf-
enamic acids inhibit calcium influx in human polymorphonuclear
Received 4 April 1997; accepted in final form 7 July 1997.
leukocytes. Mol. Pharmacol. 47: 1006–1013, 1995.
22. Kass, G. E. N., J. Llopis, S. C. Chow, S. K. Duddy, and S.
REFERENCES
Orrhenius. Receptor-operated calcium influx in rat hepato-
1. Akha, A. A., N. J. Wilmott, K. Brickley, A. C. Dolphin, A. cytes. Identification and characterization using manganese. J.
Galione, and S. V. Hunt. Anti-Ig-induced calcium influx in rat Biol. Chem. 265: 17486–17492, 1990.
B lymphocytes mediated by cGMP through a dihydropyridine- 23. Kleineke, J. W., and P. A. Janssens. Hormone-induced rise in
sensitive channel. J. Biol. Chem. 271: 7297–7300, 1996. cytosolic Ca21 in axolotl hepatocytes: extracellular origin and
2. Alvarez, J., M. Montero, and J. Garcia-Sancho. Cytochrome control by cAMP. Am. J. Physiol. 265 (Cell Physiol. 34): C1281–
P-450 may link intracellular Ca21 stores with plasma membrane C1288, 1993.
Ca21 influx. Biochem. J. 274: 193–197, 1991. 24. Kokubun, S., and H. Reuter. Dihydropyridine derivatives
3. Alvarez, J., M. Montero, and J. Garcia-Sancho. High affinity prolong the open state of Ca21 channels in cultured cardiac cells.
inhibition of Ca21-dependent K1 channels by cytochrome P-450 Proc. Natl. Acad. Sci. USA 81: 4824–4827, 1984.
inhibitors. J. Biol. Chem. 267: 11789–11793, 1992. 25. Leung, Y. M., C. F. Kwan, and T. T. Loh. Dual effects of
4. Bear, C. E. A nonselective cation channel in rat liver cells is SK&F-96365 in human leukemic HL-60 cells. Inhibition of
activated by membrane stretch. Am. J. Physiol. 258 (Cell Physiol. calcium entry and activation of a novel cation influx pathway.
27): C421–C428, 1990. Biochem. Pharmacol. 51: 605–612, 1996.
5. Berridge, M. J. Inositol trisphosphate and calcium signalling. 26. Llopis, J., G. E. Kass, A. Gahm, and S. Orrenius. Evidence for
Nature 361: 315–325, 1993. two pathways of receptor-mediated Ca21 entry in hepatocytes.
6. Berridge, M. J. Capacitative calcium entry. Biochem. J. 312: Biochem. J. 284: 243–247, 1992.
1–11, 1995. 27. Ma, Y., E. Kobrinsky, and A. R. Marks. Cloning and expression
7. Burgess, G. M., G. St. J. Bird, J. F. Obie, and J. W. Putney. of a novel truncated calcium channel from nonexcitable cells. J.
The mechanism for synergism between phospholipase C- and Biol. Chem. 270: 483–493, 1995.
adenylyl cyclase-linked hormones in liver. J. Biol. Chem. 266: 28. Mauger, J. P., J. Poggioli, and M. Claret. Synergistic stimula-
4772–4781, 1991. tion of the Ca21 influx in rat hepatocytes by glucagon and the
8. Cachelin, A. B., J. E. de Peyer, S. Kokubun, and H. Reuter. Ca21-linked hormones vasopressin and angiotensin II. J. Biol.
Ca21 channel modulation by 8-bromocyclic AMP in cultured Chem. 260: 11635–11642, 1985.
heart cells. Nature 304: 462–464, 1983. 29. Meldolesi, J., and T. Pozzan. Pathways of Ca21 influx at the
9. Catterall, W. A. Structure and function of voltage-gated ion plasma membrane: voltage-, receptor-, and second messenger-
channels. Annu. Rev. Biochem. 64: 493–531, 1995. operated channels. Exp. Cell Res. 171: 271–283, 1987.
10. Demaurex, N., A. Monod, D. P. Lew, and K. H. Krause. 30. Merritt, J. E., W. P. Armstrong, C. D. Benham, T. J. Hallam,
Characterization of receptor-mediated and store-regulated Ca21 R. Jacob, A. Jaxa-Chamiec, B. K. Leigh, S. A. McCarthy,
influx in human neutrophils. Biochem. J. 297: 595–601, 1994. K. E. Moores, and T. J. Rink. SK&F-96365, a novel inhibitor of
11. Fernando, K. C., and G. J. Barritt. Characterization of the receptor-mediated calcium entry. Biochem. J. 271: 515–522,
inhibition of the hepatocyte receptor-activated Ca21 inflow sys- 1990.
tem by gadolinium and SK&F-96365. Biochim. Biophys. Acta 31. Neher, E. Combined fura-2 and patch clamp measurements in
1222: 393–389, 1994. rat peritoneal mast cells. In: Neuromuscular Junction, edited by
12. Gögelein, H., D. Dahlem, H. C. Englert, and H. J. Lang. L. C. Sellin, R. Libelius, and S. Thesleff. New York: Elsevier,
Flufenamic acid, mefenamic acid and niflumic acid inhibit single 1989, p. 65–76.
nonselective cation channels in the rat exocrine pancreas. FEBS 32. Petersen, C. C., M. J. Berridge, M. F. Borgese, and D. L.
Lett. 268: 79–82, 1990. Bennett. Putative capacitative calcium entry channels: expres-
13. Greenwood, I. A., and W. A. Large. Comparison of the effects of sion of Drosophila trp and evidence for the existence of verte-
fenamates on Ca21-activated chloride and potassium currents in brate homologues. Biochem. J. 311: 41–44, 1995.
rabbit portal vein smooth muscle cells. Br. J. Pharmacol. 116: 33. Phillips, A. M., A. Bull, and L. E. Kelly. Identification of a
2939–2948, 1995. Drosophila gene encoding a calmodulin-binding protein with
14. Hanner, M., F. F. Moebius, F. Weber, M. Grabner, J. homology to the trp phototransduction gene. Neuron 8: 631–642,
Striessnig, and H. Glossmann. Phenylalkylamine Ca21 antago- 1992.
nist binding protein. Molecular cloning, tissue distribution, and 34. Putney, J. W. Capacitative calcium entry revisited. Cell Calcium
heterologous expression. J. Biol. Chem. 270: 7551–7557, 1995. 11: 511–624, 1990.
15. Hescheler, J., and G. Schulz. Nonselective cation channels: 35. Reinsprecht, M., M. H. Rohn, R. J. Spadinger, I. Pecht, H.
physiological and pharmacological modulations of channel activ- Schindler, and C. Romanin. Blockade of capacitive Ca21 influx
ity. Pharmacology, physiology and biophysics. In: Nonselective by Cl2 channel blockers inhibits secretion from rat mucosal-type
Cation Channels, edited by D. Siemen and J. Hescheler. Basel: mast cells. Mol. Pharmacol. 47: 1014–1020, 1995.
Birkhäuser Verlag, 1993, p. 27–43. 36. Sage, S. O., P. Sargeant, J. E. Merritt, M. P. Mahaut-Smith,
16. Hughes, B. P., and G. J. Barritt. Inhibition of the liver cell and T. J. Rink. Agonist-evoked Ca21 entry in human platelets.
receptor-activated Ca21 inflow system by metal ion inhibitors of Biochem. J. 285: 341–344, 1992.
voltage-operated Ca21 channels but not by other inhibitors of 37. Sawanobori, T., H. Takanashi, M. Hiraoka, Y. Iida, K.
Ca21 inflow. Biochim. Biophys. Acta 1013: 197–205, 1989. Kamisaka, and H. Maezawa. Electrophysiological properties
17. Hughes, B. P., K. Both, L. Harland, J. Hunt, K. M. Hurst, M. of isolated rat liver cells. J. Cell. Physiol. 139: 580–585, 1989.
Lewis, and G. J. Barritt. Identification of an mRNA species 38. Striggow, F., and R. Bohnensack. Verapamil and diltiazem
which encodes a voltage-operated Ca21 channel in rat liver inhibit receptor-operated calcium channels and intracellular
mRNA. Biochem. Mol. Biol. Int. 31: 193–200, 1993. calcium oscillations in rat hepatocytes. FEBS Lett. 318: 341–344,
18. Hughes, B. P., S. E. Milton, G. J. Barritt, and A. M. Auld. 1993.
Studies with verapamil and nifedipine provide evidence for the 39. Wallnöfer, A., C. Cauvin, T. W. Lategan, and U. T. Ruegg.
presence in the liver cell plasma membrane of two types of Ca21 Differential blockade of agonist- and depolarization-induced
inflow transporter which are dissimilar to potential-operated 45Ca21 influx in smooth muscle cells. Am. J. Physiol. 257 (Cell
Ca21 channels. Biochem. Pharmacol. 35: 3045–3052, 1986. Physiol. 26): C607–C611, 1989.C1532 INHIBITION OF CA21 INFLOW BY DIHYDROPYRIDINES
40. Wenzel-Seifert, K., D. Krautwurst, L. Musgrave, and R. Drosophila store-operated channel. Proc. Natl. Acad. Sci. USA
Seifert. Thapsigargin activates univalent- and bivalent-cation 92: 9652–9656, 1995.
entry in human neutrophils by a SK&F-I3 96365- and Gd31- 42. Zhang, J., M. Désilets, and T. W. Moon. Evidence for the
sensitive pathway and is a partial secretagogue: involvement of modulation of cell calcium by epinephrine in isolated fish hepato-
pertussis-toxin-sensitive G-proteins and protein phosphatases cytes. Am. J. Physiol. 263 (Endocrinol. Metab. 26): E512–E519,
1/2A and 2B in the signal-transduction pathway. Biochem. J. 314: 1992.
679–686, 1996. 43. Zhu, X., M. Jiang, M. Peyton, G. Boulay, R. Hurst, E.
Stefani, and L. Birnbaumer. Trp, a novel mammalian gene
41. Wes, P. D., J. Chevesich, A. Jeromin, C. Rosenberg, G. family essential for agonist-activated capacitative Ca21 entry.
Stetten, and C. Montell. TRPC1, a human homolog of a Cell 85: 661–671, 1996.You can also read
