Acetylcholine increases intracellular Ca2+ in the
rat pituitary folliculostellate cells in primary culture
Yusaku
Nakajima,
Maki
Uchiyama,
Yasumasa
Shirai,
Yasuo
Sakuma, and
Masakatsu
Kato
Department of Physiology, Nippon Medical School, Tokyo 113-8602, Japan
 |
ABSTRACT |
Pituitary
folliculostellate cells (FSCs) are thought to partially inhibit
pituitary hormone secretion through a paracrine mechanism. In this
process, one of the important questions is what factors regulate the
function of FSCs. Because ACh is synthesized in and possibly released
from the corticotrophs and lactotrophs, we examined whether FSCs
respond to ACh by the method of Ca2+ imaging in primary
cultured FSCs from male Wistar rats. ACh (30 nM-3 µM) increased
intracellular calcium concentration ([Ca2+]i)
of FSCs in a concentration-dependent manner, with an initial rapid rise
followed by a relatively sustained increase. The complete block of the
response by atropine and pirenzepine suggests involvement of muscarinic
receptors. Depletion of the stored Ca2+ by thapsigargin
blocked the response completely. Blockers of phospholipase C, U-73122
and neomycin, suppressed significantly the rise of
[Ca2+]i. These results suggest that ACh
increases [Ca2+]i in FSCs by activating
phospholipase C, presumably through activation of M1
receptors. The rise in [Ca2+]i could trigger
a variety of Ca2+-dependent cellular processes, including
the synthesis and release of bioactive substances, which in turn act on
endocrine cells.
cholinergic modulation; paracrine mechanism; calcium imaging; M1 receptor
 |
INTRODUCTION |
FOLLICULOSTELLATE
CELLS (FSCs) in the anterior pituitary do not secrete any known
traditional pituitary hormones by themselves but exert inhibitory
effects on the secretion of growth hormone (GH), prolactin (PRL),
luteinizing hormone, or adrenocorticotropic hormone (ACTH) (1,
18). FSCs share similar characteristics with glial cells and, to
some extent, with immune cells. For example, the glial protein S-100 is
a specific marker for FSCs in the anterior pituitary (15).
FSCs produce basic fibroblast growth factor (6) and
possess nitric oxide synthase (4) as well as interleukin-6 (19). Several recent reports suggest that FSCs modulate
hormone secretion through a paracrine mechanism within the anterior
pituitary (17).
To date, it is poorly understood how the activity of FSCs is regulated.
In the present study, we focused on the role played by ACh. ACh is
synthesized by corticotrophs and by a subpopulation of lactotrophs in
the anterior pituitary (3). Cholinergic modulation of GH
and PRL release in the anterior pituitary has been demonstrated (2, 11, 21). Besides, muscarinic receptors have been found in rat anterior pituitary cells; however, their precise localization remains to be resolved (14, 16). From these lines of
evidence, we hypothesized that ACh released from the endocrine
pituitary cells could increase intracellular calcium concentration
([Ca2+]i) through muscarinic receptors on the
FSCs and cause a variety of Ca2+-dependent cellular
processes. A major question here is whether FSCs express functional
cholinergic receptors and increase [Ca2+]i in
response to ACh. To test this possibility, we examined the effect of
ACh on primary-cultured FSCs by means of Ca2+ imaging.
 |
METHODS |
Primary culture of rat anterior pituitary cells.
The anterior pituitary was excised from male Wistar rats (250-300
g body wt) after decapitation by a guillotine. The pituitary was minced
and incubated in 10 ml of Dulbecco's phosphate-buffered saline (
)
(PBS, Nissui Pharmaceutical, Tokyo, Japan) containing 0.1% trypsin
(type III, Sigma, St. Louis, MO) and 0.25% collagenase (type I, Sigma)
for 20 min at 37°C with a gentle stirring. After the incubation,
pieces of the pituitary were transferred into 10 ml of PBS supplemented
with 0.1 mg/ml trypsin inhibitor (type II, Sigma) and 4 U/ml
deoxyribonuclease I (Sigma) and were dispersed by triturating with a
5-ml plastic pipette for 5 min. After washing with PBS, the cells were
plated on poly-L-lysine-coated glass coverslips and
incubated in MEM (Nissui Pharmaceutical) supplemented with 2 mM
L-glutamine, 4% normal rat serum, and 0.2% BSA (fraction V, Sigma) for 3-5 days at 37°C in a humidified atmosphere of 5% CO2-95% air. Identification of FSCs is described in
RESULTS.
Measurement of [Ca2+]i.
Details of the imaging technique and superfusion system have been
described previously (9). In brief, cultured cells were loaded by incubation with 1 µM Fura PE-3 AM (TefLabs, Austin, TX) for
60 min at 37°C. The coverslip was placed in a small superfusion chamber on the stage of a Nikon Diaphot microscope.
[Ca2+]i was recorded by using the QuantiCell
700 system (Applied Imaging, Sunderland, UK). The cells were
illuminated alternately at 340-nm and 380-nm excitation wavelengths,
and then 510-nm emission light images were captured by an
image-intensifying charge-coupled device camera (Photonics Science,
Turnbridge Wells, UK). The time interval of each 340- to 380-nm ratio
frame was 6 s. Ratios were converted to Ca2+
concentrations by the following equation (7):
[Ca2+]i = kd
[(R
Rmin)/(Rmax
R)], where
kd is the dissociation constant for Fura PE-3
Ca2+, R is the ratio, Rmin and Rmax
are the ratio values of Fura PE-3 at zero and saturating
[Ca2+]i, respectively, and
is the ratio
of fluorescence at 380 nm for Fura PE-3 in saturating and zero
[Ca2+]i.
Superfusion was performed with a control solution containing (in mM):
137.5 NaCl, 5 KCl, 2.5 CaCl2, 0.8 MgCl2, 0.6 NaHCO3, 10 glucose, 20 HEPES, and 0.1% BSA, and the pH was
adjusted to 7.4 with NaOH. Nominal Ca2+-free solution was
prepared by replacing Ca2+ with Mg2+ in the
control solution. This solution contained 10-20 µM
Ca2+ (10). Excess K+ solution was
prepared by replacing Na+ with K+ in the
control solution. The cells were continuously superfused at 37°C
throughout the experiment, and the flow rate was ~1 ml/min. All drugs
were applied through superfusion.
Drugs.
The following drugs were purchased from Wako Chemicals (Osaka,
Japan): ACh, thapsigargin, U-73343, U-73122, and pirenzepine. Nifedipine, neomycin, and ryanodine were purchased from Sigma. SKF-96365 was from Biomol Research Laboratories (Plymouth
Meeting, PA). Gadolinium chloride was from Nacalai Tesque (Kyoto, Japan).
Statistical analysis.
ACh-induced maximum changes in [Ca2+]i
(
[Ca2+]i) were considered significant when
they exceeded 10 nM from basal [Ca2+]i. At
least three independent experiments were made to draw conclusions. In
each experiment, ACh was applied twice to the same cells with a 30-min
wash period. The peak values of
[Ca2+]i in
response to the first application and to the second challenge were
designated as S1 and S2, respectively, and the
S2-to-S1 ratio (S2/S1)
was determined. In some experiments, the effects of drugs introduced
between the initial and second applications of ACh were evaluated by
frequency histograms constructed for S2/S1. The
data were expressed as means ± SD. A paired t-test was
used for statistical analysis. The significance level was set at
P < 0.05.
 |
RESULTS |
Identification of FSCs in the cultured cells.
It has been demonstrated that FSCs have distinctive morphological
characteristics in the pituitary (15). In primary culture, FSCs extended thin cytoplasm (Fig.
1A, arrows). The thin
cytoplasmic extension often formed processes in FSCs in culture. FSCs
were confirmed by immunocytochemical staining of S-100 protein, a
specific marker of FSCs in the anterior pituitary (Fig. 1B,
arrows). Ca2+ images are shown in pseudo-color (Fig. 1,
C-E). FSCs responded to 300 nM ACh (Fig.
1D) but not to 50 mM K+ (Fig. 1E). On
the other hand, non-FSCs responded to 50 mM K+ but not to
300 nM ACh.

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Fig. 1.
Ca2+ imaging of folliculostellate cells (FSCs). Arrows
indicate FSCs separated from endocrine cells in
A-E. A: phase contrast microscopy of
FSCs and other pituitary cells. B: same group of cells shown
in immunofluorescent microscopy with Cy3-labeled anti-S-100 and
FITC-labeled anti-growth hormone antibodies. C:
Ca2+ images of the same cells under resting condition.
D: Ca2+ images of the same cells observed with
300 nM ACh. FSCs increased [Ca2+]i.
E: Ca2+ images of the cells observed with 50 mM
K+. FSCs hardly responded in contrast to other pituitary
cells. It should be noted that the FSCs located at upper right
aggregated with endocrine cells. This disturbed the
Ca2+ signal of FSCs in response to ACh and to excess
K+. In this case, we excluded the response of FSCs from the
data. Scale bars in A-E, 20 µm. Color bar at
bottom indicates the intracellular calcium concentration
([Ca2+]i), 0-1,000 nM.
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The time course of the responses is shown in Fig.
2, A and B. FSCs
responded weakly to 50 mM K+ compared with non-FSCs. The
average of peak
[Ca2+]i induced by 50 mM
K+ was 42 ± 28 nM (n = 59 in 3 independent experiments) in FSCs and 646 ± 234 nM
(n = 85 in 3 independent experiments) in non-FSCs (Fig.
2C). Additionally, non-FSCs did not respond to 300 nM ACh in
these 85 cells. In the present experiment, therefore, cells were
designated as FSCs if they showed distinctive morphology and relative
unresponsiveness to excess K+ (peak
[Ca2+]i, <100 nM). We tested the validity
of these criteria in 85 cells and found that 84 of them were stained by
antisera to S-100 protein.

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Fig. 2.
Responses to excess K+ in FSCs and non-FSCs.
A: FSCs responded to 300 nM ACh but only weakly to excess
K+. B: non-FSCs responded to excess
K+ but not to 300 nM ACh. C: excess
K+-induced peak change in [Ca2+]i
( [Ca2+]i) in FSCs and non-FSCs. Each
column indicates the mean ± SD of peak
[Ca2+]i (n = 59 in FSCs,
n = 85 in non-FSCs).
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ACh-induced rise in [Ca2+]i.
Representative responses to various concentrations of ACh are shown
(Fig. 3A). FSCs responded to
ACh in a concentration-dependent manner at concentrations between 30 and 3,000 nM (Fig. 3B). No cells responded to 10 nM ACh.
However, 30 nM and 100 nM ACh elicited responses in a substantial
portion of the cells examined. Among 45 cells examined, 28 cells
responded to 30 nM ACh with peak
[Ca2+]i
of 53 ± 75 nM; 37 cells among 42 responded to 100 nM ACh with peak
[Ca2+]i of 120 ± 111 nM. All
cells examined responded to ACh at a concentration of
300 nM. The
averages of peak
[Ca2+]i induced by 300, 1,000, and 3,000 nM ACh were 229 ± 177, 301 ± 200, and
384 ± 235 nM, respectively.

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Fig. 3.
ACh-induced rise in [Ca2+]i.
A: representative responses to different concentrations of
ACh. ACh was applied for 3 min as indicated by horizontal bar.
B: values are means ± SD of peak
[Ca2+]i in no. of cells indicated beside
each symbol from 3-7 independent experiments.
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ACh elicited a rapid initial increase of
[Ca2+]i followed by a sustained phase (Fig.
4A). Similar responses were
replicated by two sequential applications of ACh at an interval of 30 min. The peak
[Ca2+]i of the first
application (211 ± 115 nM) was similar to that in the second
application (194 ± 115 nM). The histogram for the frequency
distribution of S2/S1 was ~1.0, with an
average of 0.941 ± 0.327 (n = 90 in 4 independent
experiments) as shown in Fig. 4B. This result was used as a
control for the following experiments.

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Fig. 4.
Responses to two sequential applications of ACh. A:
cells were stimulated twice with 300 nM ACh. A second stimulation,
performed after a 30-min wash period, produced a similar response. ACh
increased [Ca2+]i with an initial rapid rise
followed by a relatively sustained increase. B: the
population of the ratio of S2 to S1
(S2/S1) distributed ~1.0, and the average was
0.941 ± 0.327 (n = 90 in 4 independent
experiments).
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Muscarinic receptor antagonists.
Two muscarinic antagonists, atropine (10 nM) and pirenzepine (10 nM),
were applied 3 min before a second application of ACh. Typical examples
are shown in Fig. 5. Both atropine
(n = 54) and pirenzepine (n = 49)
abolished the ACh-induced response in all cells examined. The responses
recovered after a 30-min washout.

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Fig. 5.
Inhibition by muscarinic antagonists. A: atropine (10 nM) completely blocked the response (n = 54 in 4 independent experiments). B: pirenzepine (10 nM) also
completely blocked the response (n = 49 in 4 independent experiments). These responses were reversed after a 30-min
wash.
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Ca2+-free medium and Ca2+ channel blockers.
To determine whether an influx of extracellular Ca2+ or
mobilization of intracellular Ca2+ contributed to changes
in [Ca2+]i, Ca2+-free solution
replaced the control solution 3 min before the second application of
ACh. The Ca2+-free solution did not affect the initial rise
in [Ca2+]i but reversibly suppressed the late
sustained increase (Fig. 6A).
The peak
[Ca2+]i was 152 ± 86 nM in
the control and 134 ± 55 nM in the Ca2+-free
solution. The frequency distribution of S2/S1
remained similar to that of control, and the average was 1.002 ± 0.436 (n = 53 in 5 independent experiments), as shown
in Fig. 6B.

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Fig. 6.
Effect of Ca2+-free medium. A: removal of
extracellular Ca2+ did not affect the initial rise of
[Ca2+]i but reversibly suppressed the
following sustained increase. B: distribution of
S2/S1 of peak
[Ca2+]i was similar to control, and the
average was 1.002 ± 0.436 (n = 53 in 5 independent experiments).
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The effects of Ca2+ channel blockers are shown in Fig.
7. All blockers were applied to the bath
3 min before the second application of ACh. Gadolinium (100 µM), a
blocker of store-operated Ca2+ channels, did not affect the
initial rise of [Ca2+]i but reversibly
suppressed the following sustained increase (Fig. 7A). The
values of peak
[Ca2+]i were 220 ± 105 nM in the control and 239 ± 143 nM with gadolinium. S2/S1 distributed similarly to the control,
with an average of 1.068 ± 0.508 (n = 54 in 4 independent experiments) as shown in Fig. 7C. SKF-96365 (30 µM), a blocker of store-operated Ca2+ channels, also
reversibly suppressed the sustained increase that followed (Fig.
7B). It was noted that the initial rise was partially inhibited (P < 0.01). The peak
[Ca2+]i was 244 ± 100 nM in the
control and 181 ± 98 nM after SKF-96365. The distribution of
S2/S1 shifted to the left, with an average of
0.813 ± 0.399 (n = 59 in 4 independent
experiments) as shown in Fig. 7D. An L-type
Ca2+ channel blocker, nifedipine (10 µM), had no effect.
The peak
[Ca2+]i was 288 ± 206 nM in
the control and 318 ± 214 nM with nifedipine. No difference was
detected in the distribution of S2/S1
(n = 47 in 3 independent experiments).

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Fig. 7.
Effects of gadolinium and SKF-96365. A: gadolinium did
not block the initial rise but reversibly suppressed the following
sustained increase. B: SKF-96365 partially inhibited an
initial rise and reversibly blocked the following sustained increase.
C: distribution of S2/S1 of the peak
[Ca2+]i was similar to control, and the
average was 1.068 ± 0.508 (n = 54 in 4 independent experiments). D: distribution of
S2/S1 of the peak
[Ca2+]i was shifted to the left, and the
average was 0.813 ± 0.399 (n = 59 in 4 independent experiments).
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The contribution of intracellular Ca2+ stores was examined
by experiments with thapsigargin, an inhibitor of endoplasmic reticulum Ca2+-ATPase (8). Thapsigargin (1 µM) was
applied ~22 min before and throughout the second application of ACh.
Thapsigargin alone caused a small and transient increase in
[Ca2+]i (Fig.
8). Application of ACh, repeated after a
30-min interval, failed to induce any response in the presence of
thapsigargin (n = 60 in 3 independent experiments).

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Fig. 8.
Effect of thapsigargin. After first application, 1 µM
thapsigargin was introduced as indicated by horizontal solid bar, which
caused a slow increase of [Ca2+]i in FSCs.
The second application of ACh, performed 60 min from the first
application, did not increase [Ca2+]i
(n = 60 in 3 independent experiments).
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Phospholipase C inhibitors and ryanodine.
Inhibitors of phospholipase C, neomycin and U-73122, were used to
examine whether ACh activates phospholipase C in the FSCs. Prior
superfusion with neomycin (3 mM) for 1 h inhibited the initial rise in [Ca2+]i to 149 ± 115 nM from
the control of 246 ± 135 nM (P < 0.01), which
was partially reversed as shown in Fig.
9A. The distribution of
S2/S1 was shifted to the left with an average
of 0.617 ± 0.419 (n = 61 in 4 independent
experiments) as shown in Fig. 9D. U-73343 (10 µM, 50-min
prior superfusion), an inactive analog of U-73122, did not affect the
response (Fig. 9B). The peak
[Ca2+]i was 179 ± 117 nM in the
control and 180 ± 120 nM with U-73343. No difference was detected
in the distribution of S2/S1 as shown in Fig.
9E (n = 63 in 4 independent experiments).
U-73122 (10 µM, 50-min prior superfusion), an inhibitor of
phospholipase C, suppressed the response to 96 ± 47 nM from a
control of 177 ± 66 nM (P < 0.01), which was
reversed as shown in Fig. 9C. The distribution of
S2/S1 was shifted to the left, and the average was 0.570 ± 0.230 (n = 48 in 3 independent
experiments) as shown in Fig. 9E.

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Fig. 9.
Effects of phospholipase C inhibitors. A: neomycin (3 mM), a blocker of phospholipase C, was introduced 1 h before ACh
application and continuously superfused as indicated by horizontal
solid bar. The exact time of application is indicated in parenthesis.
Neomycin inhibited the rise of [Ca2+]i. This
inhibition was partially reversed. B: U-73343 (10 µM),
structural analog of U-73122 without inhibitory action, was applied for
50 min, as indicated in parenthesis, and washed with a control solution
for 10 min before second ACh application. U-73343 did not affect the
response. C: U-73122 (10 µM), an inhibitor of
phospholipase C, was applied in the same time course as U-73343, and it
reversibly suppressed the rise in [Ca2+]i.
D: distribution of S2/S1 of the peak
[Ca2+]i was shifted to the left by
neomycin, and the average was 0.617 ± 0.419 (n = 61 in 4 independent experiments). E: distribution of
S2/S1 of the peak
[Ca2+]i was shifted to the left by U-73122
with an average of 0.570 ± 0.230 (n = 48 in 3 independent experiments). The distribution was not affected by U-73343,
and the average was 1.066 ± 0.403 (n = 63 in 4 independent experiments).
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Ryanodine (20 µM) did not increase [Ca2+]i
of FSCs and did not affect the peak
[Ca2+]i induced by ACh. The peak
[Ca2+]i was 233 ± 130 nM in the
control and 227 ± 127 nM with ryanodine. No difference was
detected in the distribution of S2/S1
(n = 47 in 4 independent experiments).
 |
DISCUSSION |
The present experiments demonstrated that ACh increased
[Ca2+]i of FSCs in a concentration-dependent
manner. Because this response was blocked completely by atropine and
pirenzepine, the response is likely to be mediated by a muscarinic
receptor. Pirenzepine is relatively specific to the M1
receptor subtype but also blocks M4 receptors with a
similar potency (5). As we will discuss, the response of
FSCs to ACh is likely to involve an activation of phospholipase C via
Gq/11. Because M1 but not M4
receptors are coupled to Gq/11 (20), the
present action of ACh is most likely mediated by the M1 receptor.
The initial rise in [Ca2+]i was not affected
by the removal of extracellular Ca2+ but was completely
blocked by an inhibitor of the endoplasmic reticulum
Ca2+-ATPase, thapsigargin (8). These results
indicate that the initial rise in [Ca2+]i was
caused by mobilization of Ca2+ from internal stores.
However, the late sustained increase in [Ca2+]i depended completely on the presence
of extracellular Ca2+. This phase was almost completely
blocked by gadolinium, which is known to block store-operated
Ca2+ influx (12). Another inhibitor of
store-operated Ca2+ entry, SKF-96365 (13),
also caused a partial suppression. Thus the late sustained phase is
probably due to an influx of extracellular Ca2+.
Involvement of voltage-gated Ca2+ channels, however, is
unlikely, because FSCs are not excitable cells and express few of these
channels. Indeed, FSCs weakly increased the
[Ca2+]i in response to 50 mM K+.
In the present study, nifedipine, a blocker of the L-type
Ca2+ channel, did not affect the response at all. Based on
the observation that two different inhibitors of phospholipase C,
neomycin and U-73122, both suppressed the ACh-induced response, we
propose that ACh activates phospholipase C and promotes production of D-myo-inositol 1,4,5-trisphosphate in the FSCs.
It should be noted, however, that we found that U-73343, an inactive
analog of U-73122, inhibited the response to ACh when U-73343 was
present simultaneously with ACh (data not shown). This nonspecific
action of U-73343 may be due to competitive inhibition on ACh action,
because the inhibition was reversed by increasing the concentration of
ACh (our preliminary results). We did not further investigate this
point but simply washed out U-73343 before ACh application and found
that the 10-min wash completely removed the nonspecific action of
U-73343. Therefore, we applied U-73122 in the same time course as
U-73343, which must reveal a specific action of U-73122 to inhibit
phospholipase C.
An involvement of ryanodine receptors is unlikely, because ryanodine
did not increase [Ca2+]i of FSCs and did not
affect the peak
[Ca2+]i induced by ACh.
In the anterior pituitary, ACh is synthesized by corticotrophs and by a
small proportion of lactotrophs (3). ACh may be secreted
alone or with ACTH and PRL. To date, ACh has been considered to act on
somatotrophs and lactotrophs through their M2 receptors to
decrease the secretion of GH and PRL (2). The present
findings indicate that FSCs are another target of ACh action in the
anterior pituitary. The rise in [Ca2+]i in
FSCs may stimulate a variety of Ca2+-dependent cellular
processes, including synthesis and release of bioactive substances,
which in turn act on the endocrine cells.
In conclusion, ACh increased [Ca2+]i of FSCs
by activating phospholipase C through M1 receptor
activation. The present results, taken together with previous findings,
suggest that ACh could function as a paracrine factor acting on FSCs in
the anterior pituitary.
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ACKNOWLEDGEMENTS |
We are grateful to Professor V. N. Luine for critical reading
of the manuscript.
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FOOTNOTES |
This work was supported in part by Grants-in-Aid 08680872, 10670071, and 10480227 from the Ministry of Education, Science, Sports and
Culture of Japan.
Address for reprint requests and other correspondence: M. Kato,
Dept. of Physiology, Nippon Medical School, Tokyo 113-8602, Japan
(E-mail: mkato{at}nms.ac.jp).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 10 July 2000; accepted in final form 15 December 2000.
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