(Received for publication, July 27, 1995; and in revised form, January 29, 1996)
From the
Glucose stimulation raises the pH of
pancreatic
-cells, but the underlying mechanisms are not well
understood. We have now investigated the acute effects of metabolizable
(glucose and the mitochondrial substrate
-ketoisocaproic acid,
KIC) and nonmetabolizable (high K
and the K-ATP
channel blocker tolbutamide) insulin secretagogues on the pH
of pancreatic
-cells isolated from normal mice, as
assessed by BCECF fluorescence from single cells or islets in the
presence of external bicarbonate. The typical acute effect of glucose
(22-30 mM) on the pH
was a fast
alkalinization of approximately 0.11 unit, followed by a slower
acidification. The relative expression of the alkalinizing and
acidifying components was variable, with some cells and islets
displaying a predominant alkalinization, others a predominant
acidification, and others yet a mixed combination of the two. The
initial alkalinization preceded the
[Ca
]
rise associated
with the activation of voltage-sensitive Ca
channels.
There was a significant overlap between the glucose-evoked
[Ca
]
rise and the
development of the secondary acidification. Depolarization with 30
mM K
and tolbutamide evoked pronounced
[Ca
]
rises and
concomitant cytosolic acidifications. Blocking glucose-induced
Ca
influx (with 0 Ca
, nifedipine,
or the K-ATP channel agonist diazoxide) suppressed the secondary
acidification while having variable effects (potentiation or slight
attenuation) on the initial alkalinization. KIC exerted glucose-like
effects on the pH
and
[Ca
]
, but the
amplitude of the initial alkalinization was about twice as large for
KIC relative to glucose. It is concluded that the acute effect of
glucose on the pH
of pancreatic
-cells is
biphasic. While the initial cytosolic alkalinization is an immediate
consequence of the activation of H
-consuming metabolic
steps in the mitochondria, the secondary acidification appears to
originate from enhanced Ca
turnover in the cytoplasm.
The degree of coupling between glucose metabolism and Ca
influx as well as the relative efficacies of these processes
determines whether the acute pH
response of a
-cell (or of a tightly coupled multicellular system such as an
islet of Langerhans) is predominantly an alkalinization, an
acidification, or a mixed proportion of the two.
Pancreatic -cells are endocrine cells specialized in the
synthesis and secretion of insulin. Physiological release of the
hormone is the result of a complex sequence of biophysical and
biochemical events, involving entry of glucose through the GLUT-2
transporter, metabolic degradation of glucose to yield ATP, inhibition
of ATP-sensitive K
(K-ATP) channels following a rise
in the cytosolic concentration of the nucleotide, membrane
depolarization, activation of voltage-sensitive Ca
channels, rises in the cytosolic free Ca
concentration
([Ca
]
), protein
phosphorylation, and subsequent steps leading to exocytosis (for
reviews, see (1, 2, 3) ).
The study of the
modulation of cellular function by cytosolic pH
(pH) has drawn a great deal of interest in many
different cell types including pancreatic
-cells. Experimental
maneuvers thought to cause changes in the pH
of
pancreatic
-cells have long been known to affect insulin
secretion(4, 5, 6, 7, 8, 9, 10) ,
leading to the concept that glucose-evoked pH
changes might exert feedback control over the release
process(11, 12, 13) . Several effector
systems in the
-cell are either known or suspected to be
pH-sensitive in the physiological range. For example, the key
glycolytic enzyme phosphofructokinase is strongly pH-sensitive
(acidification depresses the activity of the enzyme(14) ).
Furthermore, K-ATP channel activity in
-cells is exquisitely
sensitive to pH
changes around the physiological
levels (alkalinization above resting pH
enhances
channel activity while acidification depresses
it(15, 16) ). This may in fact explain why
glucose-induced electrical activity is so sensitive to the pH
changes imposed by the administration of weak acids and
bases. Indeed, cytosolic alkalinization causes membrane
hyperpolarization and inhibition of electrical activity while cytosolic
acidification is thought to cause symmetric changes on these parameters (11, 17, 18, 19) . Interestingly, in
the absence of functional K-ATP channels (for example, in the presence
of sulfonylureas and high external Ca
concentrations), the cytosolic alkalinizing agent
NH
Cl evokes changes on bursting electrical activity which
appear to be the mirror image of those evoked under regular
conditions(20, 21) , suggesting that pH
changes affect other ion channels besides K-ATP channels in
pancreatic
-cells (for example, the L-type
voltage-sensitive Ca
channel; Ca
currents are enhanced by cytosolic alkalinization in other cell
types(22, 23) ).
Although the activation of glucose
metabolism has long been suspected to cause extensive changes in the
pH of pancreatic
-cells and of other
insulin-secreting cells(24, 25, 26) , it was
not until pH
measurements could be carried out
using intracellularly trapped fluorescent indicators (e.g. BCECF(
)(27) ) that the pattern of these changes
started to be
unravelled(9, 10, 13, 28, 29) .
Using bicarbonate-free solutions, Juntti-Berggren et al.(9) reported monophasic pH
rises in
response to glucose, which they ascribed to the activation of the
Na
/H
exchanger. In a follow-up
study(13, 30) , the authors proposed a metabolic
mechanism for the glucose-induced pH
rise in which
pyruvate transport across the mitochondrial membrane and/or oxidation
was assumed to play an essential role. In a very recent study using
bicarbonate-containing solutions, Shepherd and Henquin (29) reported sustained pH
rises in
response to glucose, which could be suppressed by DIDS, a blocker of
the Na
-dependent
HCO
/Cl
exchanger, and
strongly attenuated (although in the intermediate-high glucose
concentration range only) by replacing the external solution for a
bicarbonate-free solution. These effects were interpreted assuming
that, in the presence of bicarbonate and high glucose, the
DIDS-sensitive exchanger overcompensates and opposes an acidifying
tendency associated with Ca
influx and accumulation
in the cytosol(29) .
Using single cells and islets exposed
to a physiological (bicarbonate-containing) buffer, we have now
assessed the possibility that an acidifying mechanism linked to the
stimulation of Ca influx might contribute to the
short-term effect of glucose on the cytosolic pH of pancreatic
-cells. Furthermore, we have compared the effects of glucose and
KIC, a metabolic substrate that feeds directly the mitochondria, with
the purpose of assessing the role of mitochondrial metabolism in the
glucose-evoked pH
responses. The results indicate
that the typical acute pH
response of mouse
pancreatic
-cells to glucose is a transient rapid alkalinization,
followed by a slower acidification. While the initial alkalinization is
an immediate consequence of the activation of mitochondrial metabolism,
the secondary acidification is probably linked to enhanced turnover
(influx followed by active extrusion and/or accumulation in organelles)
of Ca
in the cytoplasm.
Figure 1:
Single islet
pH and [Ca
]
responses to glucose stimulation. A, representative
examples of single islet pH
responses to 22 mM glucose (22G), as monitored by BCECF fluorescence. B, representative examples of single islet
[Ca
]
responses to 22
mM glucose, as monitored by fura-2 fluorescence. Islets
5-8 in B are different from islets 1-4 in A. The shadowed areas denote the periods of
stimulation with high glucose. Basal glucose concentration was 3
mM.
The maximal amplitude of the alkalinizing phase (difference
between peak and basal pH), measured from experiments
displaying either multiphasic pH
responses (initial
alkalinization, followed by a pronounced acidifying phase), a pure
alkalinizing phase, or a predominant acidifying phase with residual
alkalinization in response to 22 mM glucose, was 0.11 ±
0.05 (n = 26 islets)(
); in these
experiments, the alkalinizing phase occurred 31 ± 10 s after the
beginning of the stimulus. The time-to-peak of the initial
alkalinization, measured from experiments displaying either multiphasic
pH
responses or a predominant acidifying phase, averaged 85
± 30 s (n = 20 islets).
The islets displayed
a heterogenous off response to glucose. Thus, while in most of the
islets examined, the pH was slowly and monophasically
recovered to baseline levels following the decrease in glucose
concentration from 22 to 3 mM (e.g. islet 1 in Fig. 1A), in other cases, the pH
transiently overshot the levels reached during stimulation before
it finally declined to baseline (e.g. islets 2 and 3).
Using separate islets labeled with the Ca indicator fura-2, we have characterized the effects of high
glucose on the [Ca
]
with the
main aim of assessing the physiological responsiveness of the islet
pool used for the pH
experiments. Examples of typical
responses are shown in Fig. 1B. Raising glucose
concentration from 3 to 22 mM typically resulted in a drop of
[Ca
]
below baseline lasting
1-3 min (average 88 ± 24 s, n = 12
islets). This was followed by a pronounced
[Ca
]
rise, after which the
[Ca
]
either remained elevated
(albeit with oscillations, e.g. islets 5 and 8 in Fig. 1B) or declined to lower levels (e.g. islets 6 and 7) during continued stimulation with high glucose. In
these experiments, the time-to-peak and the maximal amplitude of the
[Ca
]
responses were 270
± 58 s and 133 ± 68 nM (n = 12
islets), respectively. Removal of the high glucose stimulus was often
accompanied by a transient accentuation of the oscillatory behavior.
Due to the specificity of the available microfluorescence
detection system, the pH and the
[Ca
]
recordings were carried
out in sequence, rather than simultaneously. In the following
experiments, the cells were typically subjected to 30 mM glucose pulses for 4-6 min in the
[Ca
]
recording mode, allowed to
recover for at least 11 min in basal (2 mM) glucose, and
finally subjected to an identical high glucose pulse in the pH
recording mode. Identification of the monitored cells as
-cells relied on the specific responsiveness of the latter to
blockers of the K-ATP channel, e.g. the sulfonylurea
tolbutamide(36) . Thus, we have routinely stimulated the cells
with 250 µM tolbutamide at the beginning of each
experiment and considered a pronounced
[Ca
]
response to the
sulfonylurea as evidence that the monitored cell was indeed a
-cell (see Fig. 4B for a typical response).
Figure 4:
Relationship between depolarization-evoked
pH and [Ca
]
transients in pancreatic
-cells. A and B, representative examples of single cell pH
and [Ca
]
responses to 30 mM KCl (A) and 250
µM tolbutamide (Tolb; B), as monitored
fluorometrically from BCECF- and fura-2-loaded
-cells. Each cell
was stimulated twice with identical high K
or
tolbutamide pulses, first in the
[Ca
]
- (lighter traces
denoted by single asterisks) and then in the
pH
-recording mode (heavier traces denoted by double asterisks). Each experiment is representative of three
similar experiments. C, representative examples of single
islet [Ca
]
(islet
1) and pH
(islet 2) responses to 500
µM tolbutamide. Each experiment is representative of five
similar experiments. The shadowed areas denote the periods of
stimulation with high K
or tolbutamide. Glucose
concentration in the perifusion medium was 2 mM throughout.
Fig. 2A (traces labeled with a single
asterisk) shows that most -cells responded to the high
glucose pulse with a small and transient fall in
[Ca
]
, which was followed by a
pronounced rise toward a level several hundreds of nanomolars above
baseline. It is also apparent that both the onset and the time course
of the glucose-evoked [Ca
]
rises were highly variable from cell to cell. These
characteristics are similar to what has been described previously for
rat and ob/ob mouse
-cells(37, 38) .
Interestingly, a small fraction of the
-cells examined did not
display any [Ca
]
rises in
response to high glucose, as depicted by cell 4 in Fig. 2A.
Figure 2:
Combined pH and
[Ca
]
recordings from
single
-cells. A, representative examples of single cell
pH
and [Ca
]
responses to 30 mM glucose, as monitored
fluorometrically from BCECF- and fura-2-loaded
-cells. The shadowed areas denote the periods of stimulation with high
glucose. Basal glucose concentration was 2 mM. The cells were
stimulated twice with identical glucose pulses, first in the
[Ca
]
- (lighter traces
denoted by single asterisks) and then in the
pH
-recording mode (heavier traces denoted by double asterisks). The cells were allowed to rest in 2 mM glucose for 11-17 min (14, 15, 12, and 11 min for the
experiments depicted by cells 1 through 4,
respectively) between stimulations. B, average pH
(heavier trace denoted by a double asterisk) and
[Ca
]
(lighter trace
denoted by a single asterisk) responses to 30 mM glucose. The data were pooled from single cell experiments such as
those depicted in A. Vertical bars represent ±
S.D. of 14 single cell measurements.
The pH traces in Fig. 2A were made with a heavier line and further
labeled with double asterisks for easier identification in the
figure. In the presence of 2 mM glucose, the average resting
pH
of BCECF- and fura-2-labeled cells was 7.04 ±
0.06 (n = 14 cells). Similarly to isolated islets, the
typical pH
response of single
-cells to glucose was an
initial alkalinization of approximately 0.15 unit, followed by a slower
acidifying phase (e.g. cells 1, 2, and 3 in Fig. 2A). This pattern was representative of 58% (23
out of 39) of the cells examined. Furthermore, while some cells (34%, i.e. 13 out of 39; e.g. cell 4 in Fig. 2A) displayed a pure alkalinizing phase, others
(8%, i.e. 3 out of 39; not shown in Fig. 2A)
displayed a predominant acidifying phase with residual signs of
alkalinization. The glucose-evoked effects on
-cell pH
were often slowly reversible, usually resulting in poor recovery
within 4-6 min after withdrawing the high glucose stimulus, as
evidenced by cells 2 and 3 in Fig. 2A.
Visual
inspection of the relationship between the glucose-evoked
[Ca]
and pH
transients indicates that the initial pH
rise
occurred at a time when the [Ca
]
remained at near-basal levels following glucose stimulation (Fig. 2A, cells 1, 2, and 3; see also average data from
several single cell experiments in Fig. 2B). Indeed,
the pH
and the [Ca
]
started to rise 18 ± 7 s and 97 ± 28 s (n = 14 cells) after the delivery of the high glucose pulse,
respectively. Furthermore, the time required to raise the pH
and the [Ca
]
to half the
respective maximal levels averaged 38 ± 9 s and 209 ± 67
s, respectively. It is also apparent from Fig. 2A (cells 2 and 3) and Fig. 2B that there is a
significant overlap between the secondary acidifying phase of the
multiphasic pH
response to glucose and the rising phase of
the [Ca
]
transient, suggesting
that the acidifying response might be a consequence of Ca
influx and of the associated
[Ca
]
rise (but see the priming
data of Fig. 3and the corresponding discussion). This
hypothesis would be consistent with the response displayed by cell 4 in Fig. 2A, which is illustrative of
-cells that
failed to produce a [Ca
]
response to high glucose and did not exhibit the secondary
acidifying phase.
Figure 3:
Priming effect of glucose on
[Ca]
. The cells were
stimulated twice with 30 mM glucose in the
[Ca
]
-recording mode,
in order to assess the priming effect of the first glucose challenge.
The solution switching protocol was similar to that used in the
experiments depicted in Fig. 2. The cells were allowed to rest
in 2 mM glucose for 12-18 min (18, 16, and 12 min for
the experiments depicted by cells 1 through 3,
respectively) between stimulations. The shadowed areas denote
the periods of stimulation with high glucose. Basal glucose
concentration was 2 mM.
Closer inspection of Fig. 2A indicates, however, that in some cells (e.g. cell 3 and,
albeit to a lesser extent, cell 2) the onset of the
[Ca]
rise lags a few tens of
seconds behind the onset of the acidification. Accordingly, the
beginning of the average [Ca
]
rise does not match exactly the beginning of the average pH
fall (Fig. 2B). One possibility to interpret this
apparent discrepancy is that the cells might respond faster to glucose
once primed by a previous stimulation, as previously
acknowledged(39, 40, 41) . In order to assess
this hypothesis, we have carried out experiments whereby the cells were
subjected to the same double stimulation protocol while recording the
[Ca
]
. Under these conditions,
the operation of a priming mechanism would be expected to enhance the
[Ca
]
response to the second
high glucose challenge at earlier time points. Fig. 3shows that
this was indeed the case in most experiments. On average, the lag time
between the first and the second [Ca
]
rises was 49 ± 21 s (n = 5 cells). It is
important to note that, in these experiments, the cells were allowed to
rest for 12-18 min (average 14.5 ± 3 min, n = 5 cells) between the high glucose pulses. The
corresponding time for the combined
[Ca
]
/pH
experiments
depicted in Fig. 2was 11-17 min (average 13.5 ±
2.3 min, n = 14).
The effects of tolbutamide on
[Ca]
and pH
were
also investigated in islets that have been loaded separately with
fura-2 and BCECF, as depicted in Fig. 4C. In the later
experiments, the [Ca
]
started
to rise 27 ± 9 s (range 13-38 s, n = 5
islets) after stimulation with tolbutamide; the pH
started
to fall 36 ± 26 s (range 13-61 s, n = 4
islets) after stimulation. It is therefore possible to infer that the
pH
fall either occurs simultaneously with or is subsequent
to the [Ca
]
rise, in essential
agreement with the single cell data.
The second strategy (depicted
in Fig. 5) consisted in comparing the glucose-evoked pH responses in the presence and absence of conditions known to
suppress Ca
influx through voltage-sensitive
Ca
channels (the major modality of Ca
influx involved in glucose stimulation of
-cells (2) ). These conditions were the removal of Ca
from the extracellular medium(1) , the use of the
dihydropyridine nifedipine (a blocker of L-type Ca
channels(42) ), and the use of the hyperpolarizing agent
and K-ATP channel agonist, diazoxide (43, 44) .
Figure 5:
Modulation of glucose-evoked pH responses by the suppression of Ca
influx.
Individual BCECF-loaded islets were sequentially stimulated with 22
mM glucose, first in the absence (left) and later in
the presence (right) of the Ca
influx
suppressor (from top to bottom: exposure to a
Ca
-free solution, the L-type Ca
channel blocker nifedipine, and the K-ATP channel agonist
diazoxide). The shadowed areas denote the periods of
stimulation with high glucose. Glucose concentration in the perifusion
medium was 3 mM throughout. Each experiment is representative
of three similar experiments.
For
the experiments depicted in Fig. 5, single BCECF-loaded islets
were stimulated by raising glucose concentration from 3 to 22
mM. The major consequence of exposing the islets to
Ca-free solutions, nifedipine, or diazoxide was the
suppression of the acidifying phase of the multiphasic pH
response to glucose. It is also noteworthy that the alkalinizing
response was slower in the presence of any of these agents.
Furthermore, the steady-state pH
recorded in the absence of
Ca
or in the presence of nifedipine or diazoxide was
often (e.g. islets 2 and 3 in Fig. 4), albeit not
always (e.g. islet 1), higher than the peak pH
recorded in control.
Fig. 6A shows examples of single
-cell [Ca
]
responses to
KIC. These effects are rather similar to the homologous responses to 30
mM glucose, as can be seen by comparing the average KIC
responses depicted in Fig. 6B with the average glucose
responses depicted in Fig. 2B. It is also noteworthy
that the pH
responses of individual
-cells to KIC are
heterogenous, with some cells displaying a predominant alkalinization
and others a marked acidifying phase following an initial
alkalinization. Thus, the effects of KIC on pH
are also
qualitatively similar to the homologous responses to glucose (this
again may be better assessed by comparing the average responses
depicted in Fig. 2B and 6B). Notice however
that, on average, the maximal amplitude of the KIC-evoked initial
alkalinization is about twice as large as that of the glucose-evoked
response. Yet another difference is that, in contrast to the situation
found with glucose, we have been unable to find examples of cells with
predominant acidifying responses (and concomitant slight
alkalinizations) to KIC.
Figure 6:
Effect of the mitochondrial substrate
-ketoisocaproic acid (KIC) on the pH
and
[Ca
]
of single
-cells. A, representative examples of single cell
pH
and [Ca
]
responses to 30 mM KIC, as monitored
fluorometrically from BCECF- and fura-2-loaded
-cells. The shadowed areas denote the periods of stimulation with KIC (no
glucose present). The cells were stimulated twice with identical KIC
pulses, first in the
[Ca
]
- (lighter traces
denoted by single asterisks) and then in the
pH
-recording mode (heavier traces denoted by double asterisks). The pH
and the
[Ca
]
traces in each
panel have been superimposed for clarity. B, average
pH
(heavier trace denoted by a double
asterisk) and [Ca
]
(lighter trace denoted by a single asterisk)
responses to 30 mM KIC. The data were pooled from single cell
experiments such as those depicted in A. Vertical bars represent ± S.D. of 4 single cell
measurements.
Using physiologically buffered solutions and single
BCECF-loaded islets and isolated -cells from normal mice as a
model system to investigate the effects of glucose on cytosolic pH, we
found that the typical acute pH
response to high
(22-30 mM) concentrations of the hexose has two distinct
phases: a fast alkalinizing phase peaking at approximately 90 s and a
slower acidifying phase responsible for bringing the pH
to
near-basal levels in approximately 4 min of continued stimulation. The
presence of the secondary acidifying component has not been reported
consistently by other groups. Using monolayers of pancreatic
-cells isolated from obese hyperglycemic (ob/ob) mice and
suspensions of clonal insulin-secreting (HIT) cells exposed to
bicarbonate-free solutions, Juntti-Berggren et al.(9) reported monophasic pH
rises in response
to 8-20 mM glucose. Glucose-evoked alkalinizations of
approximately 0.16 unit had already been reported using ob/ob mouse
islets and a detection procedure based on the redistribution of
C-labeled 5,5-dimethyl-2,4-oxazolidinedione(26) ,
but the only data available from this study refers to the 7th min of
stimulation with 20 mM glucose. More recently, Shepherd and
Henquin (29) reported essentially sustained pH
rises in single pancreatic islets isolated from normal (NMRI)
mice, after long-term exposures to 7-30 mM glucose in
the presence of bicarbonate. It should also be emphasized that
multiphasic pH
responses resembling those described in our
work are already apparent from some of the single cell experiments
reported in a previous study(30) .
There has been debate in
the literature concerning the origin of the acute glucose-evoked
alkalinization in pancreatic -cells. The inhibitor of
mitochondrial pyruvate transport, 3-hydroxycyanocinnamate, has been
reported to prevent glucose-induced alkalinization, leading to the
concept that the pH
rise can either be accounted for by
pyruvate transport across the mitochondrial membrane or by its
subsequent oxidation(13, 30) . This result is
consistent with the finding that the glycolytic substrate
dihydroxyacetone increased the pH
in a glucose-like
manner(29) . We have characterized the effect of the
mitochondrial substrate KIC on the pH
aiming at clarifying
the role of mitochondrial metabolism in the glucose-evoked effect on
pH
. The fact that KIC had glucose-like effects on the
pH
indicates that the initial alkalinization induced by the
hexose is primarily the result of one or several
H
-consuming steps in the mitochondria. It is
noteworthy that KIC exceeded glucose in alkalinizing capacity, as
revealed by the larger magnitude of the initial alkalinization and by
the longer duration of the overall pH
rise (Fig. 2B versusFig. 6B). This may be
interpreted taking into account that formation of pyruvate is
associated with a net production of protons (48) .
Busa and
Nuccitelli (48) proposed that the stimulation of the aerobic
metabolism of glucose occurs at the expenses of a massive net
consumption of protons, the consequence of which would be a marked
initial rise in pH. However, the initial alkalinization
may, at a later stage, be compensated for by the cytosolic accumulation
of protons arising from ATP hydrolysis, so that in the steady-state
little or no net change in pH
may actually
occur(48) . Pancreatic
-cells might be expected to fit in
the model proposed by Busa and Nuccitelli(48) . This is
because: 1) in
-cells the aerobic transformation of glucose
largely exceeds that of anaerobic glycolysis (49) ; 2) even
assuming that pyruvate oxidation by mitochondria may not be the major
catabolic route for glucose in
-cells and that most of the
reducing equivalents are brought into mitochondria through the
operation of the glycerol phosphate
shuttle(50, 51, 52) , a net consumption of
protons would still be expected to take place due to the combined
oxidation of cytosolic NADH and electron transfer along the respiratory
chain; and 3) in stimulated
-cells, hydrolysis of cytosolic ATP is
expected to proceed at a high rate, as a consequence of enhanced
Ca
-ATPase activity (required for active
Ca
extrusion and sequestration by organelles) and
protein phosphorylation (53) .
In various cell types, the pH
set-point of the Na/H
antiporter is
raised following activation of protein kinase C with phorbol esters, a
process that might lead to pronounced increases in the
pH
(54, 55, 56) . (The prediction
of an elevated pH
applies especially to bicarbonate-free
conditions, since in the presence of the anion there might be
conflicting consequences of the activation of the
Na
/H
antiporter and of the
Na
-independent
HCO
/Cl
exchanger(57) .) The original finding that EIPA (a
blocker of the Na
/H
antiporter)
suppressed the glucose-evoked alkalinization recorded in the absence of
bicarbonate appeared to lend credit to an essential role for the
antiporter, but the fact that down-regulating protein kinase C failed
to affect the pH
rise was taken as an argument that protein
kinase C-supported phosphorylation was not involved(9) .
Nonetheless, the negative results obtained by Shepherd and Henquin (29) (dimethyl amiloride had virtually no effect on the
glucose-evoked pH
rise recorded in the presence of
bicarbonate) strongly opposes the hypothesis that activation of the
Na
/H
antiporter might mediate the
glucose-evoked alkalinization recorded under physiological conditions.
We propose the following explanation to account for the variable
effects of amiloride derivatives on the glucose-evoked alkalinization.
In resting cells, the pH
is far from equilibrium due to
sustained operation of the Na
/H
antiporter and of the Na
-dependent
HCO
/Cl
exchanger.
Blockade of the Na
/H
antiporter would
then be expected to lead to cytosolic acidification, but the actual
extent of this pH
fall depends critically on the
availability of the second exchanger to rescue the cells from the acid
load. (
)Since the key glycolytic enzyme phosphofructokinase
is thought to be markedly inhibited by modest physiological
acidifications(14) , large pH
falls (such as those
that occur in cells exposed to Na
/H
antiporter blockers in bicarbonate-free solutions) have the
potential to inhibit glucose metabolism downstream phosphofructokinase,
resulting in the suppression or pronounced inhibition of the associated
pH
rise.
Since we report that the average glucose-evoked
alkalinization is essentially over in approximately 4 min, our data are
in apparent contradiction with the study by Shepherd and
Henquin(29) , which was carried out under comparable
experimental conditions (normal mouse islets and bicarbonate-containing
solutions). It should be mentioned, however, that the sustained
pH rise reported by these authors was apparently preceded
by a transient increase lasting approximately 2.5 min. Thus, our study
concentrates on the acute effect of glucose on pH
,
whereas the latter authors address fundamentally the long-term actions of the hexose. Implicit in the model proposed by Shepherd
and Henquin (29) to account for a sustained pH
rise
is the possibility that the pH set-point of the
Na
-dependent
HCO
/Cl
exchanger
undergoes a positive shift following stimulation with high glucose
concentrations, (
)similar to what has been reported
previously to occur in other cell types in response to growth
factors(57) . The fact that the pH
rise observed
immediately after glucose stimulation is transient would then imply
that the alteration in the set-point of the exchanger has a slow time
course and only becomes noticeable by the end of several minutes of
continued stimulation.
It is well known that the secretory response
of pancreatic -cells to glucose may be enhanced by a previous
challenge with glucose or other secretagogues, in a phenomenon known as
priming, memory, or time-dependent
potentiation(59, 60) . Although the detailed mechanism
for this potentiation is not well understood, some authors have
reported evidence for the occurrence of priming at the level of
glucose-evoked [Ca
]
changes(41) . In agreement with previous
reports(39, 40, 41) , our experiments (Fig. 3) show that pretreatment of pancreatic
-cells with
30 mM glucose 12-18 min in advance to a second glucose
stimulus accelerates the latter response by an average time of 49 s.
Since the combined
[Ca
]
/pH
experiments
depicted in Fig. 2have been carried out using a double
stimulation protocol similar to that of Fig. 3(with the
[Ca
]
and pH
measurements relating to the first and second glucose pulses,
respectively), the operation of a priming mechanism may be critical to
establish an accurate relationship between the glucose-evoked
[Ca
]
and pH
changes. For example, Fig. 2B indicates that the
average pH
rise peaked 57 s after the delivery of the high
glucose pulse. Assuming that the priming mechanism affects glucose
metabolism (61) and the associated alkalinization, the
theoretical pH
transient (i.e. the pH
response to the first glucose challenge should this have been
recorded) may need to be offset by approximately 49 s compared to the
actual (recorded) response, implying that the peak of the theoretical
response may occur 106 s after the delivery of high glucose.
Analysis of the single cell experiments depicted in Fig. 2indicates that the [Ca]
started to rise, on average, 97 s after the glucose stimulus.
Thus, even taking into account the possibility of priming, our results
indicate that the initial alkalinization occurs at near-basal
[Ca
]
levels. Since the
[Ca
]
rise reflects primarily
Ca
influx through voltage-sensitive Ca
channels(32, 62) , this indicates, in agreement
with earlier reports (13, 30) , that the initial
alkalinization is not a consequence of Ca
influx.
This conclusion is further supported by the data in Fig. 5,
which show that the alkalinizing phase remained essentially intact when
the islets were stimulated with glucose in the presence of various
agents known to suppress voltage-sensitive Ca
influx (i.e. 0 Ca
, nifedipine, and diazoxide).
Interestingly, the rate of the initial alkalinization was significantly
decreased by any of these agents. This may reflect the Ca
dependence of specific mitochondrial enzymes (e.g. dehydrogenases(63) ) and suggests that Ca
concentration rises in the mitochondrial matrix are important for
the optimization of metabolic reaction rates.
We have also
demonstrated that -cells exposed to physiologically buffered
solutions undergo pronounced cytosolic acidifications in response to
glucose or KIC stimulation. We propose that this secondary acidifying
phase is specifically associated with the stimulation of Ca
influx and may be considered a consequence of enhanced
Ca
turnover in the cytosol. This is because: 1) in
the combined [Ca
]
/pH
experiments (single
-cells), the pH
decreased
concomitantly with the [Ca
]
rises after correction for priming (as seen above the predicted
lower limit for the beginning of the acidification is 106 s, in
essential agreement with the start of the
[Ca
]
rise)(
); 2) the
pH
did not decrease in cells lacking a measurable
Ca
influx in response to glucose; 3) specific removal
of Ca
influx (with 0 Ca
,
nifedipine, and diazoxide) suppressed the secondary pH
decrease. The conclusion that the stimulation of Ca
influx leads to an acidification of the cytosol is also supported
by the high K
and tolbutamide experiments, where more
direct depolarization of the cells was shown to acidify the cytosol in
essential agreement with data reported by other
authors(28, 67) . The concept that the stimulation of
Ca
influx might cause the acidification of the
cytosol was recently put forward by Shepherd and Henquin (29) to explain the finding that removal of Ca
or exposure to diazoxide strongly enhanced the pH
response recorded in bicarbonate-free medium. However, the
authors reported no effect of the Ca
influx
suppressors on the glucose-evoked alkalinization recorded in the
presence of bicarbonate.
Our experiments do not address the
mechanism by which the stimulation of Ca influx leads
to cytosolic acidifications in pancreatic
-cells. Since cytosolic
acidification appears to be a natural consequence of enhanced ATP
hydrolysis (48) and a significant fraction of the ATP yielded
by glucose oxidation is likely to be utilized by
Ca
-ATPases, the secondary acidifying component that
we found in glucose-stimulated
-cells may reflect primarily
H
accumulation associated with active Ca
transport out of the cells and into internal stores.
Alternatively, Ca
accumulated intracellularly may
displace protons from binding sites in the cytosol and/or exchange for
protons when it is incorporated in organelles (e.g. endoplasmic reticulum), as proposed for other cell
types(68, 69, 70) .
In conclusion, we have
shown that acute glucose stimulation of pancreatic -cells evokes a
multiphasic pH
response consisting of an initial
alkalinization and a secondary acidification. Underlying these two
phases are essentially distinct but interlinked mechanisms. While the
initial alkalinization is linked to the activation of
H
-consuming metabolic steps in the mitochondria, the
secondary acidification is probably linked to enhanced Ca
turnover in the cytosol (Ca
influx and
Ca
extrusion/sequestration).