Vol. 280, Issue 6, R1688-R1696, June 2001
Heat acclimation and heat stress have different effects on
cholinergic-induced calcium mobilization
Pavel
Kaspler and
Michal
Horowitz
Division of Physiology, Faculty of Dental Medicine, and
Department of Physiology, The Hebrew University, Jerusalem 91120, Israel
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ABSTRACT |
There is evidence that
the signal transduction array responsible for the secretion of water in
evaporative cooling by the submaxillary gland of the rat is subject to
heat acclimatory responses. The objectives of the present study were
1) to examine whether heat acclimation affects intracellular
Ca2+ mobilization and, in turn, submaxillary glandular
responsiveness; 2) to assess whether the acclimatory
responses differ from those evoked on heat stress (HS). Experiments
were conducted on submaxillary glands of rats acclimated at 34°C for
0, 2 [short-term heat acclimation (STHA)], and 30 [long-term heat
acclimation (LTHA)] days. The resting cytosolic calcium concentration
([Ca2+]c) and the carbamylcholine-evoked
calcium signal ([Ca2+]s) of dispersed
glandular cells were measured using the fluorescent dye fura 2 AM.
Inositol-1,4,5-trisphosphate (IP3)-sensitive endoplasmic reticulum Ca2+ stores were determined in permeabilized
cells using fura 2 potassium salt. STHA resulted in a drop in both
[Ca2+]s and IP3-sensitive
Ca2+ stores. On LTHA, the [Ca2+]s
amplitude reverted to the preacclimation value, whereas the IP3-sensitive Ca2+ stores remained low. The
drop in [Ca2+]s on STHA is in accord with the
decreased glandular output (measured by 86Rb efflux)
observed during this acclimation phase. However, after LTHA the
enhanced glandular output despite reduced
[Ca2+]s levels suggests an increased
efficiency of cellular secretory mechanisms in that group.
Collectively, the alterations in [Ca2+]s
support our biphasic acclimation model (Horowitz M, Kaspler P, Marmari
Y, and Oron Y. J Appl Physiol 80: 77-85, 1996.).
In nonacclimated glands, HS caused an elevation in
[Ca2+]s coincidentally with a decrease in the
IP3 Ca2+ stores. In contrast,
[Ca2+]s in both STHA and LTHA glands was not
affected by HS, despite a marked increase in the
IP3-sensitive Ca2+ stores in the LTHA glands.
The opposing responses to HS and heat acclimation in calcium signaling
and stores confirm the specificity of each process.
muscarinic signaling; salivary gland; evaporative cooling; intracellular calcium
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INTRODUCTION |
HEAT ACCLIMATION
INCREASES the sensitivity and efficiency of the evaporative
cooling system (26, 32). Namely, less oxygen is consumed
per volume of water secreted, and there is less secretion per larger
wetted area (32). Furthermore, at a similar stimulation frequency, the heat-acclimated gland produces, over time, a larger secreted volume than that before acclimation. These changes are attributable to cellularly localized adaptations, e.g., membrane receptor density or affinity and hyperplasia and hypertrophy, elicited
by temporally varying neurohumoral and intracellular responses during
the process of heat acclimation (15, 17).
In the rat salivary gland, as well as in the sweat glands of many other
species, the major pathway for water secretion in evaporative cooling
is via muscarinic signaling. This pathway has been extensively studied
with respect to heat stress and heat acclimation in the submaxillary
salivary glands of the acclimating rat model. Horowitz et al.
(15) showed that short-term heat acclimation (STHA)
upregulates the density of the muscarinic receptors (MR) and lowers
their affinity. An altered ratio of low-affinity to high-affinity MR
subtype populations was also observed together with changes in the
magnitude of the calcium signal evoked by supramaximal concentrations
of carbamylcholine (CCh). Under similar conditions, in the rat parotid,
Fujinami et al. (7-9) reported an array of transient
cellular responses involving the upregulation of MR, coincident with
the reduced generation of inositol-1,4,5-trisphosphate (IP3), a second messenger in the MR transduction cascade,
and a drop in Ca2+ mobilization in response to
IP3 administration. This was followed by a decrease in
carbachol-stimulated IP3 generation and CCh-evoked calcium
concentration signal ([Ca2+]s) amplitude. In
contrast to STHA, long-term heat acclimation (LTHA) has been less
studied. We know only that on LTHA, further upregulation of MR density
occurs, increasing to preacclimation affinity (13, 15).
Collectively, the temporal biochemical changes, which have been
measured in several isolated gland preparations, are time correlated
with impaired glandular responsiveness during STHA, followed by the
resumption of normal glandular function together with increased
sensitivity and responsiveness on LTHA. The evidence accumulated thus
far leads us to hypothesize that increased glandular sensitivity is
achieved through greater receptor affinity, whereas augmented capacity
is achieved through increased receptor density or glandular size.
In contrast to heat acclimation, heat stress either has no effect on,
or downregulates MR and increases the affinity of the MR, in both
salivary glands and in a salivary gland cell line, HSY. Stevenson et
al. (35) and Calderwood et al. (3) reported elevated IP3 levels and increased calcium influx in
response to heat stress in several cell lines, i.e., an opposite
response to that deduced for acclimation homeostasis. Collectively,
these findings confirm differences between the heat stress effects and the acclimatory response.
In the MR signaling pathway, cytosolic calcium signaling provides an
important mediation component triggering many cellular processes.
Hence, the aim of the present study was twofold: 1) to
examine whether heat acclimation affects intracellular Ca2+
mobilization and, in turn, the responsiveness of the rat submaxillary salivary gland; 2) to assess the specificity of heat
acclimation vs. heat stress specificity. For this purpose, the quantity
of the calcium present in the endoplasmic reticulum and calcium signals were measured and analyzed with respect to the physiological
functioning of the salivary glands of heat-acclimated rats. We also
studied the calcium signals during heat stress superimposed on heat
acclimation. Our data show that the endoplasmic reticulum
IP3 calcium stores and the calcium signals are subject to
thermal adaptation.
Cumulatively, our findings indicate that heat acclimation reduces both
the evoked calcium signal and the cellular calcium stores, despite an
enhanced secretion in the LTHA phase. This may suggest that acclimation
leads to an increased efficiency of cellular processes distal to the
calcium signal. Heat stress differed from heat acclimation by
increasing the evoked calcium signal, suggesting that heat acclimation
is a specific thermal response. On acclimation, acclimatory adaptations
blunt the heat stress effect.
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MATERIALS AND METHODS |
Heat acclimation and heat stress.
Male rats (Rattus norvegicus, Sabra strain, albino var.;
Harlan, Jerusalem) weighing 200-250 g were used. The animals were assigned to normothermic (controls, C) or heat-acclimated (AC) groups.
All animals were kept under a 12:12-h light-dark cycle. C rats were
kept at 24 ± 1°C, whereas heat acclimation was achieved by
continuous exposure to 34 ± 1°C and 30-40% relative humidity, for 2 days (STHA) or 30 days (LTHA) (14). Each group was
further subdivided into rats that did not undergo any further treatment and those subjected to heat stress at 41 ± 1°C for 2 h. To
assess the effectiveness of the treatments, the rectal temperature of the animals was measured using rectal thermistors (YSI 402; Yellow Springs, OH) inserted 6 cm beyond the anal sphincter (13,
15).
The basal cytosolic calcium level ([Ca2+]c),
the [Ca2+]s, and the
IP3-sensitive calcium stores were measured in all the
experimental groups. Our previously published database characterizing
the physiological features of the acclimated submaxillary gland in
response to CCh stimulation was used to establish the
[Ca2+]s-glandular function correlation. All
experiments were performed in accordance with guidelines approved by
the Hebrew University Committee for Animal Experimentation.
Dispersed cell preparation.
Animals were killed by cervical dislocation. The submaxillary glands
from each animal were quickly excised, thoroughly minced, and incubated
in 10 ml of digesting medium consisting of Krebs bicarbonate buffer (in
mM: 117 NaCl, 4.2 KCl, 1.2 KH2PO4, 1.2 MgCl2, 24 NaHCO3, and 1 g/l glucose, pH 7.4)
containing 5.4 U collagenase and 20 µU hyaluronidase and were aerated
with a mixture of 95% O2-5% CO2 and shaken
for 1 h at 37°C. The suspension was homogenized each 10 min by
10 passages through a Pasteur pipette. The nondigested debris was
discarded, and the suspension was centrifuged (10 g, 1 min)
to remove the digesting medium. The pellet was then washed twice and
resuspended in Krebs bicarbonate buffer containing 2.5 mM
CaCl2 and 1% BSA (fraction V, Sigma).
Resting Ca2+ level in dispersed
glandular cells.
[Ca2+]c was measured for the salivary gland
cells of each experimental rat group immediately after termination of
the dispersed cell preparation. Likewise,
[Ca2+]c was measured in the presence of
gradually elevated extracellular concentrations of Ca2+ to
get some indications on the capacity of the cell membrane of the
various groups to regulate [Ca2+]c at
different external calcium loads. Salivary gland cell suspensions were
loaded with fura 2 AM (6 µM) at 37°C with continuous aeration and
shaking, as before, for 30 min, then washed and resuspended in fresh
Krebs containing calcium (2.5 mM) but without BSA. The cells were
washed free of fura 2; the pellet was washed twice in calcium-free
Krebs, containing 10 mM EGTA, and resuspended in the same medium.
Resting [Ca2+]c was determined by
establishing a calcium dose-response curve for resting
[Ca2+]c. The external calcium solution was
prepared from a 250 mM stock Ca2+ solution added to the
experimental cuvette stepwise under constant stirring to establish a
dose-response curve within a range of 0-2.5 mM. The
[Ca2+]s was measured using a fluorimetric
ratio system (PTI). To determine the actual free Ca2+
concentration obtained at each dilution point, free Ca2+
levels were measured before the experiments using the ratiometric fura
2 K salt as the fluorescent dye (see below).
[Ca2+]c was measured at each external
Ca2+ concentration. The maximal response was calculated by
fitting to the dose-response curve.
CCh-evoked
Ca2+ signal in dispersed
glandular cells.
The cell suspension was loaded with fura 2 AM as before. The cell
pellet was resuspended in 18 ml of the same Krebs solution and divided
into six aliquots (3 ml) to obtain a dose-response curve for the
muscarinic agonist CCh (0.5-300 µM). [Ca2+] was
measured as above.
Intracellular Ca2+
store responsiveness in permeabilized cells.
The Ca2+ store responsiveness was measured according to
Falsafi et al. (6) with some modifications. Cell
suspensions from salivary glands were prepared as above. The cells in
the final pellet were permeabilized by incubation in 25 mg/l saponin
for 2.5 min at room temperature as determined in preliminary
experiments using trypan blue. The permeabilized cells were washed
three times in Krebs buffer and resuspended in 3 ml Krebs containing
180 nM Ca2+, an ATP regenerating system (20 mM creatine
phosphate, 10 U/ml creatine phosphatase, 3 mM MgATP), 10 µg/ml
oligomycin, and 10 µM antimycin A to prevent substrate oxidation. The
cells were incubated in this medium for 30 min at 37°C with
continuous shaking. Fura 2 K salt was then added to the cell suspension
to a final concentration of 2 µM.
Release of Ca2+ from the stores was evoked by the specific
second messenger IP3 added four times cumulatively to raise
the evoking concentration in the range of 1-18 mM. The amount of
released Ca2+ was normalized for cell number (mg protein in
the measured suspension volume). For this purpose, aliquots of the cell
suspension were taken before the signal measurement, lysed with 0.1 M
NaOH, and protein was determined according to Bradford.
[Ca2+] was measured as above.
Glandular output.
To characterize the [Ca2+]s-submaxillary
glandular performance relationship, the correlation between the
CCh-evoked [Ca2+] signals obtained in this investigation
and glandular performance expressed by specific markers in response to
an equimolar CCh stimulation was determined. For this purpose, we
resorted to previous databases on dose-response curves for CCh-evoked
86Rb efflux, a marker of CCh-MR coupling (15)
and CCh-induced salivation (22). Briefly, 86Rb
efflux was measured in a perifusion system using gland slices loaded
with 86Rb. For stimulation, slices were superfused with
aerated (95% O2-5% CO2) Krebs buffer
containing CCh. 86Rb efflux was measured in the outflow
fractions (30). Salivary flow was measured in rats with a
chronically cannulated submaxillary gland. For glandular stimulation,
the drug was administered as a bolus via the jugular vein.
Unfortunately, in the latter experiments, a complete series was
available only for nonacclimated and 2 days-acclimated rats
(22). In all the experimental series, both glandular
physiological performance and [Ca2+]s were
measured at the same CCh stimulation range.
Calcium quantification, calculations, and statistics.
Cytosolic calcium was calculated from the ratio of the fluorescence
emitted at 510 nm at the two excitation wavelengths, 340 and 380 nm,
using 161 nM as the dissociation constant (Kd)
of fura 2 for Ca2+ at 25°C, the temperature at which all
measurements were conducted. The Kd of fura 2 for Ca2+ at this temperature was derived by extrapolation
from Grynkiewicz et al. (11) and Haugland
(12). The calcium concentration was determined at the end
of each individual dose-response curve by measuring the limiting
340/380 ratio for the unbound fura form in free calcium medium and the
limiting ratio for the bound form at maximal calcium concentration
subsequent to cell permeabilization with saponin at the end of each
experiment. In the suspension of permeabilized cells, the maximal
signal was measured in the presence of 10 mM Ca2+, and the
minimal signal was measured by gradual adding of 0.5 M EGTA stock
solution to the experimental cuvette. Both the saturating Ca2+ concentration for the limiting bound form ratio and
the EGTA concentration measured for the limiting ratio of the unbound
fura were determined by stabilization of the peak signal and its
absolute quenching, respectively.
All dose-response curves were approximated by a generalized
Michaelis-Menten model (5), where the external agent at
the different experimental series was CCh, IP3, or external
Ca2+. The maximal response to the agonist [maximal
response amplitude (MRA)] was obtained by fitting the dose-response
curves [modified Lineweaver-Burk plot of the dose-response curve
(5)]. The calculated MRA for each individual treatment
was used to calculate responses as a percent of the maximal response.
To assess significant changes, ANOVA, Student's t-test, or
nonparametric statistics were used. P < 0.05 was
considered significant.
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RESULTS |
Resting Ca2+ level in
the course of heat acclimation.
There was no significant change in the resting
[Ca2+]c in acclimated vs. nonacclimated rats,
as reflected in the calcium dose-response curves of the resting
glandular cells and in the basal [Ca2+]c
measured in the CCh dose-response experimental series. However, the
basal [Ca2+]c measured in the latter
experimental series was lower than that measured at a similar (2.5 mM)
external calcium concentration in the calcium dose-response experiments
(Table 1). The only statistically
significant difference among the groups was the coefficient of the
equation fitting the dose-response curves, which in the control rats
was markedly lower than unity (0.58) and in the LTHA group increased to
0.78.
CCh-evoked
Ca2+ signal and
IP3 endoplasmic reticulum
Ca2+ stores.
As shown in Fig. 1, the response to CCh
stimulation was dose dependent. In the nonacclimated group, the maximal
response was beyond the range of the concentrations used. In contrast,
during STHA, the maximal CCh-evoked response peaked at 100 µM CCh,
and this was 26% lower than in the controls (P < 0.02). On LTHA, the [Ca2+]s amplitude
returned toward preacclimation level. The MRA was lower than in the
nonacclimated group by 19%. No significant changes in EC50
were observed among the groups (Table 2).
An example of individual records of the time course of the
Ca2+ on CCh addition is provided in Fig.
2. Figure 3
shows the IP3 receptor-mediated pool size. Heat acclimation
decreased the IP3 receptor-mediated release of calcium from
the endoplasmic stores, both at STHA and LTHA. However, the curves
presenting the values as percent MRA (Fig. 3, bottom) show
right and left shifts in the STHA and LTHA groups, respectively. The
EC50 for these groups (Table 2) differed significantly
(P < 0.05), suggesting differences in the sensitivity
of the response to IP3 between the STHA and the LTHA
groups.
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Fig. 1.
Carbamylcholine (CCh)-evoked Ca2+
concentration ([Ca2+]) signal in dispersed cells of the
submaxillary gland of rats undergoing short- and long-term heat
acclimation (STHA and LTHA, respectively). The data represent the
absolute [Ca2+] signal amplitude. Values are presented as
the means ± SE, n = 10 or 11. Significant
difference from the nonacclimated group: *P < 0.05. Inset: the [Ca2+] signal evoked by 10 µM CCh
in the absence of extracellular Ca2+ is presented. C,
control; d, day.
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Fig. 2.
Time course of the [Ca2+] signal on CCh addition in
dispersed cells of the submaxillary gland of nonacclimated and STHA and
LTHA rats. Maximum and minimum ratio values were 11.7 and 0.81, respectively.
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Fig. 3.
Inositol-1,4,5-trisphosphate (IP3)-sensitive
[Ca2+] stores in permeabilized dispersed cells from the
submaxillary gland of rats undergoing STHA and LTHA. Top:
absolute [Ca2+] values; bottom: signal values,
expressed as percentage of the maximal response amplitude (MRA). For
MRA values, see Table 2. The data are presented as the means ± SE, n = 5-12. Significant difference from the
nonacclimated group: **P < 0.0005, *P < 0.01/P < 0.05.
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Glandular performance:
[Ca2+]s-glandular
output relationships.
Data from our previous studies (16, 22) allow us to
correlate glandular performance with the evoked
[Ca2+]s, an essential step in the
mobilization of water secretion. The
[Ca2+]s-CCh-stimulated 86Rb
efflux (a marker for agonist-receptor coupling) relationship is
demonstrated in Fig. 4. It is evident
that during STHA, at the lower [Ca2+]s range
(corresponding to a lower CCh dose), the 86Rb efflux was
much smaller than in the control glands. The maximal [Ca2+]s obtained in this group corresponded
to supramaximal CCh stimulation and induced an 86Rb efflux
similar to that of the control at a similar
[Ca2+]s. However, the STHA glands, as opposed
to the C glands, were unable to produce greater signals to further
increase secretion. LTHA exhibited a biphasic curve: at the lower
[Ca2+]s range, a very low 86Rb
efflux was observed. The high-amplitude signals were associated with an
86Rb efflux markedly exceeding that of the C gland at
similar signal amplitudes. This may suggest two distinct receptor
populations at this acclimation phase, with a more efficient response
in at least one of the receptor populations.
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Fig. 4.
Effects of parasympathomimetic-induced
[Ca2+]s on submaxillary glandular performance
in acclimated rat. Top: 86Rb efflux-evoked
intracellular [Ca2+] signal relationship;
bottom: pilocarpine-induced saliva flow. Data were compiled
from Refs. 15 and 22, respectively, and this
investigation, and are presented as the means ± SE,
n = 7-9.
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Agonist-induced saliva flow was used as an estimate of glandular
secretory processes distal to [Ca2+]s (Fig.
4). The available data are only partial. However, similar to the
[Ca+2]s-86Rb efflux relationship,
during STHA, greater [Ca+2]s increments were
required to produce similar saliva flows. Unfortunately, such data are
not available for the LTHA phase.
Heat stress superimposed on heat acclimation.
Acute heat stress reduced resting [Ca2+]c in
the LTHA glands when external Ca2+ was gradually raised
(Fig. 5). The MRA of the
[Ca2+]c in the LTHA cells subjected to
increasing calcium concentrations was 109.1 ± 2.4 nM in the LTHA
group vs. 150.1 ±11.6 nM in the C group. The right shift of the curve
showing the values as percent MRA (Fig. 5, bottom) indicates
decreased sensitivity of the LTHA cells to external calcium loads.
During heat stress, in nonacclimated glands CCh-evoked
[Ca2+]s was markedly higher than in the
nonheat stress-matched group (Fig.
6), and EC50 was
significantly higher (8.9 ± 0.7 vs. 2.95 ±0.63, 0.01 < P < 0.05, for nonacclimated heat-stressed vs.
nonacclimated glands, respectively). In contrast, the acclimated cells
did not differ significantly from the nonstressed cells (not shown).
IP3-sensitive stores dropped significantly in the
nonacclimated groups. In the LTHA glandular cells, the
IP3-sensitive store was markedly elevated with a
concomitant decrease in the sensitivity of the response to
IP3 stimulation compared with the nonheat stress condition in this group (Fig. 7). The difference in
EC50 for the two groups was significant (LTHA-HS: 6.66 ±2.37; LTHA 3.23 ±1.24; P < 0.05).
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Fig. 5.
Resting [Ca2+] level in dispersed
cells from the submaxillary gland of heat-acclimated, heat-stressed
rats during external calcium load. Top: absolute cytosolic
[Ca2+] ([Ca2+]c) values;
bottom: signal values, expressed as percentage of the MRA.
For MRA, see Table 1. The data are presented as the means ± SE,
n = 5-8. Significant difference from the matched
nonheat-stressed groups: ***P < 0.0005, **P < 0.005. Inset: [Ca2+]
dose-response curves for the nonheat-stressed groups are presented. It
is noteworthy that heat stress unmasks a significant difference between
the LTHA and the nonacclimated groups.
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Fig. 6.
CCh-evoked [Ca2+] signal in dispersed cells
of the submaxillary gland of heat-stressed rats undergoing STHA and
LTHA. Top: absolute [Ca2+] signal amplitude;
bottom: signal values, expressed as percentage of the MRA.
Inset: C vs. C-heat stress (HS) are compared. For MRA
values, see Table 2. The data are presented as the means ± SE,
n = 10-13. Significant difference from the
nonacclimated group: ***P < 0.0005, **P < 0.00.5, *P < 0.05. Significant
difference from the heat-stressed control group: ###P < 0.0005.
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Fig. 7.
IP3-sensitive [Ca2+] stores in
permeabilized dispersed cells from the submaxillary gland of
heat-stressed rats undergoing STHA and LTHA. Top: absolute
[Ca2+] values; bottom: signal values,
expressed as percentage of the MRA. For MRA, see Table 2. Significant
difference from the matched nonstressed groups: *P < 0.01/0.05.
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DISCUSSION |
Our present results support our previous studies indicating that
the muscarinic signaling for water secretion undergoes temporal changes
during heat acclimation and the amplitude of the evoked [Ca2+]s is the target for both chronic
and acute thermal effects. During heat acclimation,
[Ca2+]s undergoes biphasic changes, and the
mechanisms leading to these changes differ in the two studied phases.
During the STHA phase, [Ca2+]s is reduced
coincidentally with a drop in glandular output. On LTHA, the
high-amplitude [Ca2+]s (Fig. 4) induces a
greater glandular response than in nonacclimated glands, suggesting
that processes other than calcium mobilization contribute to this
beneficial effect. These responses are heat acclimation specific and
override opposing heat stress-induced effects such as augmented
CCh-induced [Ca2+]s, which, in terms of the
agonist-mediated signal, are only seen in the nonacclimated rats.
Resting calcium level.
The basal calcium level in the resting exocrine gland is subject to
changes via alterations in both the cell membrane and the intracellular
stores (e.g., 10, 21, 25, 31, 34). Although there were no significant
differences between the various experimental groups, the contribution
of the controlling mechanism varied somewhat because they appeared to
have different kinetics, as inferred from Table 1. These mechanisms are
beyond the scope of our investigation (it is noteworthy that the
resting [Ca2+]c of cells exposed to
progressively elevated calcium concentrations was higher than that of
cells maintained at 2.5 mM calcium throughout the experiments). We
hypothesize that this phenomenon stems from an altered load on the
plasma cell-membrane pumps in the two experimental conditions.
Calcium signals.
In the present investigation, the size of the IP3-sensitive
calcium pool size and the magnitude of the membrane receptor-mediated calcium signal were measured. The qualitative effects of heat acclimation on these variables are presented in Table
3. During the transient STHA phase,
CCh-evoked [Ca2+]s was markedly reduced with
no change in the sensitivity of the response. Concomitantly,
IP3-sensitive endoplasmic stores were significantly
reduced, together with a decrease in the sensitivity of the response to
IP3. Previous studies reported decreased affinity of the MR
to the agonist on STHA (15), due, at least in part, to the
effects of acclimation on the receptor-coupled G protein (19). This finding may localize the basolateral cell
membrane as the first site at which, during STHA, impairment in the
muscarinic transduction pathway takes place, leading to diminished
IP3 production and decreased release of calcium from the
endoplasmic stores. Several steps in this cascade were reported by
Fujinami et al. (9) in the parotid gland.
On LTHA, CCh-evoked [Ca2+]s tended to revert
to the preacclimation level. At this acclimation stage, upregulation of
MR with preacclimation affinity had taken place (15).
Because there was no rise in [Ca2+]s, it was
suggested that the drop in IP3-sensitive calcium stores (compared with the nonacclimated condition) blunts the increase in
[Ca2+]s amplitude to levels exceeding that of
the nonacclimated signal. This conclusion is supported by the finding
that the CCh-evoked [Ca2+]s at zero
extracellular calcium level (Fig. 1, inset) diminished by
the same order of magnitude in the C and the LTHA groups, suggesting that the contribution of extracellular calcium to the evoked
[Ca2+]s (4, 18, 24, 27, 29, 34)
is similar for both groups. It is noteworthy that the left shift of the
IP3 dose-response curve for the LTHA group may imply
increased sensitivity of the response to IP3, allowing the
observed faster response on agonist stimulation.
In conclusion, it is likely that during STHA, decreased MR affinity and
reduced IP3-sensitive calcium pool size contribute to the
smaller STHA [Ca2+]s, whereas recovery of the
[Ca2+]s on LTHA depends mainly on alterations
in the plasma membrane. Temporal changes in unsaturated free fatty
acids and free cholesterol levels on heat acclimation, leading to
altered plasma membrane properties and function, were recently measured
in salivary glands of short- and long-term heat-acclimated rats (H. Shmida, P. Kaspler, M. Horowitz, and Y. Barenholz, unpublished
observations). These changes may account for the resumed
preacclimation affinity of the MR in the LTHA glands.
The evocation of [Ca2+]s activates
K+ and Cl
channels in the basolateral and
luminal cell membranes, respectively. The result is a net loss of these
ions, water mobilization into the acinar lumen, and cell shrinkage. The
correlation between [Ca2+]s and the efflux of
K+, Cl, or salivary flow may shed light on
the polarity of the acclimatory responses in this water secretion
pathway and indicate whether processes other than
[Ca2+]s amplitude are responsible for
glandular function in the course of heat acclimation. The relationship
between [Ca2+]s and K+ marker
efflux, which signifies receptor activation, as well as the
[Ca2+]s-saliva flow relationship (Fig. 4),
implies that at equimolar CCh stimulation there is no correlation
between the amplitude of the [Ca2+]s produced
and glandular performance among the nonacclimating and acclimating
groups. At the range of the higher [Ca2+]s
produced in LTHA glands, the glandular
output-to-[Ca2+]s ratio was greater in the
LTHA glands than in the nonacclimated glands for similar
[Ca2+]s, suggesting increased efficiency of
processes distal to the [Ca2+]s on acclimation.
STHA glands fail to produce high-amplitude
[Ca2+]s. In this group, the slope of the
[Ca2+]s-saliva flow relationship is similar
to that of the control, implying that the decreased output is
associated, at least partially, with lowered
[Ca2+]s. This may be associated with
decreased CCh-induced IP3 production or decreased
IP3-induced response. Our previous studies (e.g., Ref.
15) indicated lower MR affinity. However, because there was no change in EC50 in the CCh dose-response curves, it
is likely that during STHA IP3 production is the
rate-limiting step in glandular performance.
Effect of heat stress.
During heat stress, the acquired heat acclimation advantage of the LTHA
cells is the avoidance of elevated cytosolic calcium. This was
manifested in the present investigation by the reduced basal
[Ca2+]c and by its attenuated rise with an
increasing calcium load. This fits many other observations from our
laboratory on altered features of the cell membrane in heat-acclimated
glands as well as in cardiomyocytes (H. Shmida et al., unpublished
observations) possibly blunting stress-induced calcium overload (M. Horowitz and R. Shlomai, unpublished observations).
On glandular stimulation, heat stress accelerates Ca2+
influx and mobilizes intracellular calcium stores, leading to a rise in
intracellular Ca2+ level due to unsynchronized inhibition
of adenylate cyclase and phosphatydilinositol-2-phosphate signaling
pathways (1, 2, 3, 20). Our data on the heat-stressed
control group are in accord with these findings, as manifested by the
marked elevation in the amplitude of the
[Ca2+]s. This elevation can be partially
explained by the marked increase in MR affinity (15),
possibly leading to augmented IP3 production, as shown for
heat-stressed cells by several investigators (35) and or
by increased activity of the Na/Ca exchanger, as shown for human
endothelial cells (23). It does not tally,
however, with the observed decrease in the IP3-sensitive
calcium pool, implying the contribution of extracellular calcium to the
signal produced. Collectively, glandular performance, as indicated by 86Rb efflux (15), was impaired.
In contrast to the nonacclimated heat-stressed rats, the
heat-acclimated heat-stressed groups did not differ in their CCh-evoked [Ca2+]s from the nonstressed, heat-acclimated
rats. Different mechanisms, however, contributed to this apparent
stability in the two acclimation phases. In the STHA glands, neither of
the factors studied differed from those of the nonstressed matching
group, suggesting that changes occurring during the STHA state override
the effects of heat stress. In contrast, on LTHA, despite implications
of augmented IP3-sensitive Ca2+ stores, the
sensitivity of the IP3 response decreased. Qualitative presentation of changes in MR signaling before and after heat stress
with heat acclimation is presented in Table
4.
View this table:
[in this window]
[in a new window]
|
Table 4.
Qualitative presentation of changes in muscarinic receptor signaling
before and after heat stress with heat acclimation
|
|
Taken together, our present data, as well as that from a previous study
(15) and unpublished observations (P. Kaspler and M. Horowitz), suggest that heat acclimation and heat stress produce a
diametrically opposing effect on the MR signaling cascade. In nonacclimated rats, the effects of heat stress are primarily
intracellular and downstream of the MR. On acclimation, however,
adaptive changes take place both in the plasma membrane MR and
intracellularly. These confer protection against the deleterious
effects of heat stress on calcium homeostasis. Interestingly, such
changes, particularly with respect to calcium turnover, have been
observed in thermotolerant cell lines as well (20).
Perspectives
Calcium homeostasis is a major factor in cellular integrity. Our
findings that calcium signaling provides a target for long-standing acclimatory responses engender new concepts and merit a large-scale study of calcium compartmentalization (e.g., caffeine-sensitive stores)
and calcium trafficking and its regulation. The possibility of
molecular changes leading to altered expression of the targeted proteins involved in calcium signaling is still an open question as
well. Studies from our laboratory on calcium regulation in the heart
support this notion.
| |
FOOTNOTES |
Address for reprint requests and other correspondence: M. Horowitz, Dept. of Physiology, Hadassah Medical School, The Hebrew Univ., POB 12272, Jerusalem 91120, Israel (E-mail:
horowitz{at}cc.huji.ac.il).
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 28 September 2000; accepted in final form 2 February 2001.
| |
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