(Received for publication, August 22, 1994; and in revised form, December 21, 1994)
From the
Intact mouse islets were loaded with
2`,7`-bis(2-carboxyethyl)-5(6)-carboxyfluorescein to study the effects
of glucose on cytoplasmic pH (pH) in pancreatic
B-cells. In HCO
buffer, glucose produced
a steady-state increase in pH
that required
metabolism of the sugar and was concentration-dependent between 0 and
10 mM (K
5 mM)
before plateauing at a maximum value of
0.2 pH units. In HEPES
buffer, glucose concentrations above 7 mM caused an initial
rise followed by a secondary decrease and an eventual return to about
initial values. Inhibition of Ca
influx had little
effect on the pH
changes produced by glucose in
HCO
medium, but unmasked an alkalinizing
effect in HEPES buffer. Raising cytoplasmic Ca
by 30
mM potassium caused a larger acidification in HEPES than in
HCO
buffer, but a subsequent rise in
glucose now increased pH
in both types of buffer.
In the presence of 4,4`-diisothiocyanostilbene-2,2`-disulfonic acid
(DIDS; inhibitor of
HCO
/Cl
exchange), the
effect of glucose on pH
in
HCO
buffer became similar to that in
HEPES buffer. After inhibition of the Na
/H
exchanger by dimethylamiloride, glucose produced a marked and
sustained fall in pH
in HEPES buffer. A similar
fall was seen in HCO
buffer only when
DIDS and dimethylamiloride were present together. However, if
Ca
influx was prevented when both exchangers were
blocked, glucose increased pH
. In conclusion, the
metabolism of glucose tends to increase pH
in
B-cells, whereas the concomitant rise in
[Ca
]
exerts an
acidifying action. In HEPES buffer, this acidifying effect of
Ca
is offset by the operation of the
Na
/H
exchanger. In physiological
HCO
buffer, the activity of the
HCO
/Cl
exchanger
overcompensates and leads to an increase in pH
.
Glucose plays a pre-eminent role in the control of pancreatic
B-cell function(1) . The mechanisms by which it stimulates
insulin release involve regulation of a number of ionic events through
changes in B-cell
metabolism(1, 2, 3, 4, 5, 6) .
The major events can be summarized as follows. Glucose entry in B-cells
is followed by an acceleration of glycolysis and glucose oxidation,
which generates signals that close ATP-sensitive K channels in the plasma membrane. The resulting decrease in
K
conductance leads to depolarization with subsequent
opening of voltage-dependent Ca
channels.
Ca
influx through these channels increases, causing a
rise in free cytoplasmic calcium
[Ca
]
, (
)which serves as the triggering signal for the exocytosis
of insulin granules. The metabolism of glucose also augments insulin
release by amplifying the effectiveness of
[Ca
]
on the secretory
machinery(7) .
It has been speculated that protons might be
one of the signals produced by glucose metabolism and that changes in
cytoplasmic pH (pH) in B-cells might influence
certain steps of stimulus-secretion
coupling(8, 9, 10, 11) . Therefore,
several studies have examined the effect of glucose on B-cell
pH
, but rather contradictory results have been
obtained probably because of the use of different preparations (whole
islets, dispersed islet cells, tumoral cell lines), of different
methods of pH
measurement, and of different
buffers. The weight of the evidence, however, indicates that glucose
produces a slight alkalinization of
B-cells(12, 13, 14, 15, 16) .
On the other hand, the mechanisms by which this alkalinization might
occur are not established(11) . The current hypothesis that it
is brought about by an activation of the
Na
/H
exchanger with overcorrection of
the acidifying action of glucose metabolism (16) has not taken
into consideration the possible contributions of
[Ca
]
and of the
HCO
/Cl
exchanger.
In
this study, intact pancreatic islets from normal mice were loaded with
the pH-sensitive dye BCECF and examined by microspectrofluorometry. Our
aim was to monitor pH in islet cells over longer
periods of time than in previous studies, during stimulation by various
concentrations of glucose in the presence and absence of
HCO
, Ca
, and inhibitors
of the Na
/H
and
HCO
/Cl
exchangers.
The perifusion
chamber was mounted on the stage of an inverted microscope (Nikon
Diaphot) used in the epifluorescence mode with a 20 objective.
BCECF was successively excited at 440 and 490 nm by means of two narrow
band-pass filters mounted on a computer-controlled motorized filter
wheel placed in front of a 75-watt xenon lamp. A dichroic mirror
centered at 510 nm reflected the UV light to the perifusion chamber and
transmitted the emitted fluorescence, which then passed through another
filter of 535 nm. Fluorescent images were obtained with a CCD video
camera (Photonic Science Ltd., Tunbridge Wells, UK) at a resolution of
256
256 pixels. They were then digitized into 256 gray levels
and analyzed with the MagiCal system (Applied Imaging). To improve the
signal-to-noise ratio, eight consecutive 40-ms frames were averaged at
each wavelength before ratioing. The time interval between successive
series of 440-490 images varied according to the length of the
experiment, being 3.5 s over 15 min and 7.0 s over 30 min. Hence, the
period of exposure to the excitatory light was the same irrespective of
the duration of the experiment. The pH
was calculated from
an in vitro calibration curve constructed from the ratio
values obtained by perifusing solutions of different pH values (ranging
between 5 and 9.5) containing 1.8 µM BCECF-free acid (the
concentration giving a similar intensity of signal as that of
BCECF-loaded islets). The medium had the following composition: 136
mM KCl, 4 mM NaCl, 5 mM MgCl
, 5
mM glucose, and 20 mM HEPES. Bovine serum albumin was
omitted to prevent its precipitation at high pH values.
Basal islet cell pH values (in 3 mM glucose) averaged 6.98 ± 0.01 (n = 189) and
6.95 ± 0.01 (n = 134) in
HCO
and HEPES buffers, respectively. This
small, statistically significant (p < 0.05) difference was
observed in all experimental series, as will be seen in the figures. It
is, however, important to emphasize that these values correspond to
steady-state pH
. Sudden omission of
HCO
/CO
by changing to HEPES
buffer was followed by a rapid increase in pH
before the
secondary decrease below initial values. Conversely, changing from
HEPES buffer to HCO
buffer initially
caused a transient acidification (data not shown). Qualitatively
similar observations have been made with single mouse
B-cells(20) .
Figure 1:
Influence of various glucose
concentrations, of mannoheptulose, and of dihydroxyacetone on
pH in mouse islets. BCECF-loaded islets were
perifused with HCO
medium (solidlines) or HEPES medium (brokenlines). A-C, the concentration of glucose (G) was 3
mM at the start of the experiments and was then changed as
indicated on top of each panel. D, the concentration of
glucose was raised from 3 to 15 mM in a medium containing 20
mM mannoheptulose (MH) throughout. E,
dihydroxyacetone (DHA) was added to a medium containing 3
mM glucose. Each trace is the mean ± S.E. for nine
islets.
Figure 4:
Influence of the omission of extracellular
Ca on pH
in mouse islets.
BCECF-loaded islets were perifused with HCO
medium (solidline) or HEPES medium (brokenline). The concentration of glucose (G) was
increased from 3 to 15 mM after 5 min. Ca
was omitted, and 50 µM EGTA added between 15 and 25
min. The traces are the means ± S.E. for 9 and 10 islets,
respectively.
In the absence of HCO, the decrease in
pH
that followed the omission of glucose from the
perifusion medium (Fig. 1A) was again larger (p < 0.01) than that occurring spontaneously when the islets were
perifused with a medium containing 3 mM glucose throughout. A
small monophasic rise in pH
was observed on the change from
3 to 7 mM glucose (Fig. 1B). When the glucose
concentration was raised to 10 mM and above, islet pH
changed in three phases: a transient rise was followed by a
decrease and an eventual return to about initial values (Fig. 1C).
Fig. 2illustrates the
concentration dependence of the glucose-induced rise in islet
pH. The measurements were made at steady state, 15 min
after the change from 3 mM glucose to a medium containing
another glucose concentration. They are presented as absolute pH
values (Fig. 2A) or as the change in pH
within the same islet (Fig. 2B). In a
HCO
buffer, the relationship was
hyperbolic with a K
of
5 mM and a
maximum at
15 mM glucose. In the absence of
HCO
, the relationship was more complex.
pH
increased between 0 and 7 mM glucose, while the
size of the pH
rise became smaller at higher glucose
concentrations (Fig. 2B).
Figure 2:
Concentration dependence of the effects of
glucose on pH in mouse islets. pH
was measured in experiments similar to those shown in Fig. 1. A, pH
at 19 min, i.e. 15 min after changing the glucose concentration; B,
change in pH
relative to the control value in the
same islet at the start of the experiment (in glucose at 3
mM). Values are the means ± S.E. for 7-13 islets
perifused with HCO
or HEPES medium as
indicated.
Because of the distinct
effects of high glucose concentrations on pH in islets
perifused with HCO
or HEPES medium, we
checked that the discrepancy was not due to local alkalinization of the
HCO
medium by CO
loss in the
open perifusion chamber. In the first series, the flow rate of the
HCO
solution was doubled, but this did
not prevent 15 mM glucose from increasing pH
by
0.10 ± 0.02 units compared with 0.11 ± 0.01 units at the
usual flow rate of 1.4 ml/min. In the second series, 5 mM HEPES was added to the HCO
buffer.
Under these conditions, 15 mM glucose still increased pH
by 0.10 ± 0.01 units. These values are not statistically
different.
Diazoxide opens ATP-sensitive
K channels without interfering with metabolism and
thereby prevents glucose from depolarizing B-cells and from increasing
[Ca
]
(17, 22) . When the concentration of glucose was
increased from 3 to 15 mM in the presence of diazoxide, a
sustained rise in pH
was observed not only in
HCO
buffer, but also in HEPES buffer (Fig. 3A).
Figure 3:
Influence of B-cell membrane potential on
pH in mouse islets. BCECF-loaded islets were
perifused with HCO
medium (solidlines) or HEPES medium (brokenlines). A, diazoxide (Dz) was added to the medium before the
concentration of glucose (G) was raised from 3 to 15 mM to prevent the depolarization of B-cells. B,
extracellular potassium was raised to 30 mM at the time of
diazoxide addition to depolarize B-cells. The concentration of glucose
was then raised from 3 to 15 mM in the presence of high
potassium and diazoxide. The traces are the means ± S.E. for
9-12 islets.
When the B-cell membrane was depolarized
and cytoplasmic [Ca]
was
increased by 30 mM potassium in the presence of 3 mM glucose and diazoxide (7) , a fall in pH
occurred (Fig. 3B). This fall was much larger in
HEPES than in HCO
buffer. A subsequent
rise in the glucose concentration to 15 mM was followed by a
sustained rise in pH
in both types of solutions (Fig. 3B). Under these conditions, the rise in glucose
does not affect steady-state
[Ca
]
(7) .
Omission
of extracellular Ca during stimulation with 15 mM glucose markedly lowers [Ca
]
in islet cells(17, 23) . This did not result in
any major change in pH
when the islets were perifused with
HCO
buffer, but caused a marked
alkalinization in HEPES buffer (Fig. 4). Ca
reintroduction into the medium was followed by an acidification
only in HEPES buffer.
Figure 5:
Influence of blockers of the
HCO/Cl
and
Na
/H
exchangers on the pH
changes induced by glucose in mouse islets. BCECF-loaded
islets were perifused with HCO
medium (solid lines) or HEPES medium (broken lines). Ten
min before the concentration of glucose (G) was raised from 3
to 15 mM, the following substances were added to the medium:
40 µM DMA (A), 200 µM DIDS (B), and 40 µM DMA and 200 µM DIDS (C). These substances remained present until the end of the
experiment. The traces are the means ± S.E. for 9-12
islets.
As expected,
DIDS did not affect pH in HEPES buffer, when no
HCO
/Cl
exchanger is
operative. DIDS was also without effect in HCO
buffer containing 3 mM glucose, but profoundly modified
the changes in islet pH
induced by 15 mM glucose (Fig. 5B). These became similar to those observed in
HEPES medium without DIDS (compare with Fig. 1C).
The combination of DMA and DIDS produced essentially similar effects
in HCO and HEPES buffers (Fig. 5C). There occurred a decrease in pH
in 3 mM glucose, and the rise in glucose concentration
to 15 mM produced a small initial increase in pH
followed by a marked decrease.
Finally, we evaluated the
contribution of [Ca]
to the
fall in pH
caused by glucose when both
Na
/H
and
HCO
/Cl
exchangers are
inhibited. The [Ca
]
rise
normally produced by glucose was prevented by diazoxide (17) or
by blocking Ca
channels with nimodipine(24) .
When diazoxide was present in HCO
medium
supplemented with DIDS and DMA or in HEPES medium containing DMA,
pH
no longer decreased, but increased following the rise in
the glucose concentration from 3 to 15 mM (Fig. 6). An
increase in pH
was also produced by 15 mM glucose
in HEPES medium supplemented with DMA and nimodipine (data not shown).
The acidification of islet cells, produced by glucose when the
Na
/H
and
HCO
/Cl
exchangers are
inhibited, can thus be ascribed to the rise in
[Ca
]
that glucose causes.
Figure 6:
Role
of Ca in the pH
changes induced by glucose in mouse islets after blockade of
the HCO
/Cl
and
Na
/H
exchangers. BCECF-loaded islets
were perifused with HCO
medium (solidline) or HEPES medium (brokenline).
Ten min before the concentration of glucose (G) was raised
from 3 to 15 mM, the following substances were added to the
medium: 40 µM DMA, 200 µM DIDS, and 250
µM diazoxide (Dz) in
HCO
buffer and 40 µM DMA and
250 µM diazoxide in HEPES buffer. These substances
remained present until the end of the experiment. The traces are the
means ± S.E. for 7-10 islets.
Figure 7:
Effects of glucose on insulin release
from mouse islets. Batches of 20 islets were perifused with
HCO medium (
) or HEPES medium
(
). The concentration of glucose (G) was raised from 3 to
15 mM as indicated. In B and C, the medium
was supplemented with 40 µM DMA and 200 µM DIDS, respectively. Note that the perifusion system is different
from that used for pH
measurements. Values are
means ± S.E. for four to five paired
experiments.
Intact pancreatic islets, which contain 80%
insulin-secreting B-cells, were loaded with BCECF and perifused for
25-30 min with both HCO
and HEPES
buffers to study the transient and sustained changes in pH
brought about by glucose. In previous studies, static systems, a
single type of buffer, and/or dispersed pancreatic cells have generally
been used for relatively short periods of time(11) .
The
existence of Na/H
exchangers in islet
cells is established, but the presence and possible function of
HCO
/Cl
exchangers are
less clear (review in (11) ). Experiments using the ammonium
prepulse technique (27) showed that both types of exchangers
contribute to the recovery from an imposed acid load in mouse B-cells. (
)This study further shows that both the
Na
/H
and
HCO
/Cl
exchangers also
participate in the control of basal pH
. When the islets
were perifused with HCO
medium containing
3 mM glucose, pH
was little affected by separate
blockade of the exchangers. A stronger acidification occurred when DMA
and DIDS were combined in HCO
buffer or
when DMA was added to HEPES buffer, in which DIDS alone was without
effect, as expected. We therefore felt it important to compare the
effects of high glucose concentrations in HCO
buffer (when both exchangers are operative) and in HEPES buffer
(when the HCO
/Cl
exchanger is not operative).
Glucose metabolism in B-cells leads to closure
of ATP-sensitive K channels, membrane depolarization,
and rise in [Ca
]
. This study
shows that the alkalinizing effect of glucose does not depend on
closure of ATP-sensitive K
channels and B-cell
depolarization because it persisted when both events were prevented by
diazoxide (5, 22) and was not reproduced by
tolbutamide, which depolarizes B-cells by closing ATP-sensitive
K
channels(5, 22) . The
alkalinization is also independent of the membrane potential itself
because it occurred when glucose was added to both polarized B-cells
(4.8 mM potassium + diazoxide) and depolarized B-cells
(30 mM potassium + diazoxide). Finally, the rise in
pH
produced by glucose is not secondary to the
[Ca
]
rise because it occurred
under conditions where glucose is known not to increase
[Ca
]
(4.8 mM potassium
+ diazoxide) (17) or to only transiently decrease an
already elevated [Ca
]
(30
mM potassium + diazoxide)(7) . Omission of
extracellular Ca
was also without significant effect.
It should be noted, however, that the rise in pH brought
about by 15 mM glucose did not display a biphasic pattern when
glucose could not also raise
[Ca
]
. Recent experiments have
shown that the rise in [Ca
]
lags slightly behind the rise in pH
in single
B-cells(28) . Further experiments will be necessary to
determine whether it is involved in this biphasic change.
We also
wish to point out that, before starting the pH experiments, the quality
of all islet preparations was assessed by measuring glucose-induced
[Ca]
changes. Oscillations in
[Ca
]
similar to those
previously reported (17) were consistently seen. On the other
hand, no oscillations in pH
were detected even when the
data acquisition rate was identical to that at which
[Ca
]
oscillations could easily
be identified.
We cannot exclude the possibility that
pH
oscillations escaped detection with our technique or
could not be seen in a multicellular preparation because they are
asynchronous in different cells.
Omission of extracellular Ca from a medium
containing 15 mM glucose, which results in a fall in
[Ca
]
(17, 23) ,
was followed by a prompt rise in pH
, which attained values
similar to those measured in HCO
buffer.
Moreover, when the glucose-induced
[Ca
]
rise was prevented with
diazoxide, a rapid sustained alkalinization was caused by 15 mM glucose. This alkalinization also occurred in the presence of high
potassium and diazoxide, when glucose does not increase the already
elevated [Ca
]
(7) .
Finally, the relationship between glucose concentration and pH
in HEPES and HCO
buffers started to
diverge above 7 mM, i.e. above the threshold for
membrane depolarization and stimulation of Ca
influx(1, 4, 5) . The stronger
acidifying effect of [Ca
]
in
HEPES compared with HCO
buffer is also
evidenced by the larger fall in pH
produced by 30 mM potassium in the former buffer.
Our results therefore show that
the genuine effect of glucose is similar in the absence or presence of
HCO. The difference in the pH
changes results from the masking of the alkalinization by a
strong [Ca
]
-induced
acidification that is normally prevented by the
HCO
/Cl
exchanger.
Further support for this interpretation is provided by the experiments
using blockers of the exchangers (see below). That the
HCO
/Cl
exchanger is
somehow activated in the presence of high glucose is compatible with
the observations that glucose stimulates HCO
uptake by (29) and Cl
efflux from (30) islet cells and that a fraction of this stimulation is
Ca
-dependent, even though the interpretation of this
dependence is not straightforward.
The important role of the
HCO/Cl
exchanger is
strikingly demonstrated by the effects of DIDS. This blocker of the
exchanger (33, 34) was without effect in HEPES buffer,
when the exchanger is not functioning because of the absence of
HCO
. On the other hand, in
HCO
buffer supplemented with DIDS, the
response to glucose became similar to that occurring in HEPES buffer.
After a transient rise, pH
decreased slightly before
returning to levels similar to those measured before the concentration
of glucose was raised. This stabilization of pH
at steady
state is obviously achieved by the Na
/H
exchanger. Thus, when both exchangers were inhibited, either by
combination of the pharmacological blockers or by addition of DMA alone
to HEPES buffer, the initial small increase in pH
produced
by 15 mM glucose was followed by a marked acidification.
To
determine whether this acidification is a proper effect of glucose or
is the consequence of the rise in
[Ca]
, the latter was prevented
with diazoxide or nimodipine. Under these conditions, glucose increased
pH
. It is thus clear that the genuine effect of glucose is
to increase pH
, not to decrease it, when the
Na
/H
and
HCO
/Cl
exchangers are
not operative. The decrease in pH
is caused by the rise in
[Ca
]
that glucose also
produces.
In conclusion, contrary to previous assumptions, the
metabolism of glucose in islet cells tends to increase pH,
whereas it is the rise in [Ca
]
occurring in glucose-stimulated B-cells that exerts an acidifying
action. In HEPES buffer, the acidifying effect of
[Ca
]
is offset by the operation
of a Na
/H
exchanger, so that after
transient fluctuations, pH
returns to approximately basal
values at steady state. In physiological HCO
buffer, the activity of the
HCO
/Cl
exchanger
overcompensates and leads to the increase in pH
.