Camp. Biochem. Physiol. Vol. 94A. No. 4, pp. 673-676, 1989 0300-9629/89 $3.00 + 0.

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Printed in Great Britain ‘CT1989Pergamon Press plc

LONG-TERMHEAT ADAPTATION RESULTS IN AN

ENHANCED EFFICIENCY OF
MUSCARINICALLY-INDUCED WATER SECRETION IN
RAT SUBMAXILLARY GLANDS

YORAM ORON,* OFER FALACH, IZCHAK MARMARY and MICHALHOROWITZ?

Division of Physiology, Hadassah School of Dental Medicine, The Hebrew University, Jerusalem 91010,
Israel; and *Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv
University, Ramat Aviv, Israel

(Received 12 May 1989)

Abstract-l. Carbamylcholine-induced @‘Rb+ and 16C1- efflux, as markers of calcium mobilization and
water secretion, respectively, were studied during 30 days of heat acclimation (at 34°C) in rat submaxillary
gland slices using perifusion techniques.
2. The fractional rate of ‘6CIm efflux was markedly elevated with acclimation, reaching its maximal level
on day 30, while that of 86Rb+, after an initial rise, returned to non-acclimated control levels. The total
carbamylcholine-induced efflux of both ions markedly increased throughout the 30 days’ acclimation.
3. The rapid increase in ion fluxes was accompanied by a transient increase in Na+ concentrations in
the gland and a decrease in the saliva.
4. The data suggest that the acclimation-induced increase in secretory capacity is bi-phasic: initially,
a rapid transient rise in ion fluxes accompanies a transient rise in muscarinic receptor density (Kloog et
al., 1985).
5. Long term acclimation is characterized by increased efficiency of the cellular secretory mechanism(s),
as demonstrated by the chronically increased efflux of ions.

INTRODUCTION changes in glandular secretory capacity, we studied

carbamylcholine (CCh)-induced efflux of 86Rb+ and
Chronic exposure of rats to moderate heat results in 36C1- as markers for cellular calcium mobilization
the development of various adaptive mechanisms and the resulting trans-cellular water secretion. Na+
leading to increased tolerance during heat stress. and K+ concentrations in the gland and in the saliva
The emergence of heat-adapted animals is a were also measured. Our results suggest that follow-
dynamic process, characterized by clearly defined ing initial decrease in glandular responsiveness, which
phases at different organ levels. The rat’s is compensated for by increased central cholinergic
submaxillary salivary gland, the major effector stimulation (Horowitz and Meiri, 1985), the long
organ for evaporative cooling, has been exten- term adaptation of the gland to heat is brought about
sively studied in this respect. Heat acclimation by the increased efficiency of the cellular secretory
brings about an increased capacity for secretion pathway.
and an increased efficiency of the glandular appar-
atus (Horowitz and Argov, 1978; Horowitz et al.,
MATERIALSAND METHODS
1983). In the intact animal, the initial period
of acclimation is characterized by augmented Male Rams noruegicus (Zabar strain, albino variation) of
saliva flow rate, due to a transient increase in both 250-280 g initial body weight, were acclimated for 2-30 days
gland’s neural stimulation and density of its by continuous exposure at 34 + 1’C and 3545% R.H.
muscarinic receptors. All of the above phenomena are Matched control rats were maintained at 24 + 1-C. All
coincidental with a transient decrease in glandular animals were supplied with food and water ad libirum
responsiveness (Horowitz et al., 1984; Horowitz and (Horowitz, 1976). Animals were sacrificed by cervical
dislocation and submaxillary glands were excised. Gland
Meiri, 1985; Kloog et al., 1985). With progression of
slices were pre-incubated for 2040min in Krebs-Ringer
heat acclimation, the density of muscarinic receptors bicarbonate solution (KRB) under O&O, (95:5)
and neural activity, both return to control level. atmosphere with constant shaking at 37”C, in the presence
Nevertheless, the capacity for increased salivary of 5 or lOpCi/ml of 86RbCl or Na36C1, respectively.
secretion is retained (Horowitz and Meiri, 1985; Portions, equivalent approximately to a quarter of a gland,
Kloog et al., 1985; Horowitz et al., 1983) suggesting were taken for efflux measurements. For 86Rb, glandular
the emergence of post-receptor mechanisms of tissue was continuously perifused with O,/CO, (95:5)-
adaptation. equilibrated KRB at 37°C and aliquots were collected every
To investigate the cellular basis of these complex 60 sec. For WI efflux, KRB at 24°C with 12 set sampling
time were employed. Preliminary experiments have shown
that at 37”C, using the technique described for 86Rb, the rate
tAuthor to whom all correspondence should be addressed. of 36C1 efflux was too rapid to follow. However, increased

673
674 YoRhM ORoN et al.
sampling rate and decreased temperature (to 24°C) allowed
for reproducible j6C1 efflux determinations. The unstimu-
lated effiux rates were established by a 10 min (for 86Rb) or
2min (for 36Cl) perifusion with KRB alone. Immediately
after, 10 u M CCh was added to the nerifusion solution. At
the end of each experiment, residual*labei was measured in
the slice homogenates. Results were calculated as maximal
fractional rates of efflux [(label in each fraction/residual
label at that time) x lOO].The instantaneous residual label
was calculated by a computer programme from final
residual value and the radioactivity content in the individual
fractions (Nadler et al., 1986). For total efflux calculations,
In residual label was plotted vs time of perifusion and the
first order constant of efflux was calculated. The addition of
CCh to the perifusate transiently increased the rate of efflux.
Total efflux (% of total label) was calculated as a difference
between the extrapolated and the actual residual label.
Typical efflux profile of CCh-stimulated *6Rb efflux is shown
in Fig. 1.
Na+ and K+ concentrations in glandular tissue
homogenates and in the saliva were measured by flame
photometry, as previously described (Horowitz et al., 1978).
For statistical analysis, Student’s t-test at P < 0.05
significance level was used.
RESULTS
In control rats, the basal (unstimulated) fractional
rates of 86Rb were much lower than the correspond-
ing fractional rates of 36C1 efflux (2.7 +0.2 vs
22.1 + 2.0% per min, respectively). Heat acclimation
did not result in consistent changes in the basal
fractional rates of efflux of either ion (Table 1). This
implies that the basal activity of K + and Cl- channels
is not affected by the acclimation process. There were,
however, pronounced changes in the CCh-stimulated
fractional rates of efflux and the total efflux of both
ions. On day 2 of acclimation both the duration and
the maximal fractional rate of 86Rb efflux were higher
than in slices of control glands. As a result, there was
4.7
1 the chorda tympani innervating this gland (Kloog et
0 10 20
TIME (MN)
3.6
0 2 4 6 8 10 12 14 16 18 20
TIME (MIN)
Fig. I, Measurements of ion fluxes in submaxillary slices: the
figure shows the first order plot of the rate of 86Rb efflux in
a representative control experiment. The inset shows frac-
tional rates of efflux (mean + SE) of six control experiments.
a significant enhancement of the fraction of total
glandular 86Rb released by exposure to CCh. On days
5 and 30 of acclimation, the fractional maximal rates
of efflux returned to control values. The duration of
the stimulated efflux was, however, longer than in
control slices. Hence, the fraction of 86Rb released by
CCh was significantly higher in acclimated animals
throughout the entire heat exposure period
(Fig. 2A, B). The maximal fractional rate of ‘6C1
efflux also increased on day 2 of acclimation without
a significant change in the duration of the response.
This pattern was observed also on days 5 and 30 of
acclimation (Fig. 2A, B). Thus, similarly to the CCh-
induced response of 86Rb flux the fraction of 36CI
released by CCh was elevated throughout the
acclimation period.
The marked changes in the CCh-induced fractional
rates of K and Cl efflux rates may have affected the
tissue balance of Na+ and K+ ions. We have,
therefore, measured the saliva and the glandular
content of both ions in control and acclimated
animals. Heat acclimation resulted in a transient
increase in glandular Nat (by 50% on day 5 of
acclimation) and a concomitant decrease in saliva (by
55% on day 5), without a significant change in both
glandular and salivary concentrations of K+. Upon
prolonged acclimation, the concentrations of Na+ in
the submaxillary glands and in the saliva returned to
control values (Table 2).
DISCUSSION
The present report provides evidence for intracellu-
lar changes leading to increased efficiency of the
cellular secretory mechanism in the submaxillary
salivary gland upon prolonged heat acclimation. Our
data show that total efflux of both 86Rb and 36C1
increased despite unchanged density of glandular
muscarinic receptors and decreased neural activity of
al., 1985; Horowitz and Meiri, 1985).
The current model for glandular water secretion
postulates that receptor activation results in the
recruitment of calcium leading to opening of K+
channels on the basal and of Cl- channels on the
apical membrane of the acinar cell. The increased Cl-
efflux into the lumen provides the driving force for
water secretion (Petersen, 1988). Our observation of
rapid increase in both 86Rb+ and 36C1- efflux
concomitantly with the increase in the density of
cholinergic receptors (Kloog et al., 1985) implies that,
initially, the increased capacity for secretion is pro-
vided by a transient receptors recruitment. Prolonged
exposure to heat, however, is characterized by a
return of receptor density to control level (Fig. 2C.
Kloog et al., 1985). The continuous requirement for
increased secretory capacity during heat acclimation
D appears therefore to be satisfied by increased
efficiency of the muscarinic transduction processes.
This increased efficiency is exhibited in the present
22
investigation by two different effects on K+ and Cl-
channel activity. The increase in *6Rb efflux is effected
by prolonging the response period, whereas elevated
36CI efflux is determined by the maximal fractional
efflux rate. The doubling of 36C1/86Rbmaximal efflux
rate ratio at the end of the acclimation period
 Water secretion in submaxillary glands 675

0 10 20 30

TIME (DAYS)

Fig. 2. (A) *6Rb and ‘6C1maximal fractional efflux rates basal fractional rate was subtracted and (B) total
CCh-induced efflux, presented as % increase of control, with (C) correlation to the density of muscarinic
receptors. For calculations see Fig. I and Materials and Methods. Each point represents the mean value
obtained from six to seven animals. Vertical lines denote SE; asterisks denote significant difference from
control (day 0): *P < 0.05, **P -c0.01, ***P< 0.001. The changes in muscarinic receptor density was
calculated from Kloog ef al. (1985).

Table I. Basal efflux (M i SE) and carbamylcholine-induced 36CI-

to s’Rb+ efflux ratio in submaxillary gland slices of heat acclimated
Table 2. Gland and saliva Na+ and K + concentrations (M _+SD)
rats
in heat acclimated rats
Fractional rate
Gland Saliva
basal efflux Ratio of max
(%/min) fractional rate Acclimation Nat Kf Na’ K+
Acclimation of CCh-induced (Days) (me&) (meqil)
(Days) “Rb+ 36CI “‘Cl- /86Rb’ efflux’
C 27.0 + 4.0 87.0 f 4.0 10.3 * 2.1 44.8 f 2.0
C 2.7 f 0.2 22.1 f 2.0 0.96 2 39.0 f 1.0’ 105.0 + 5.0. 4.0 f 0.8’ 37.8 f I .5
2 2.9 f 0.1 28.9 i 3.1 1.16 5 42.0 i 1.0” 91.0 f 10.0 5.7 * 1.2’ 40.1 + 2.1
5 2.4 k 0. I 20. I * I .8 1.17 I2 33.0 * 1.0 92.0 + 8.0 14.0 + 3.3 39.6 + 2.9
30 2.8 f0.1 26.7 + 3.0 2.12
‘0.01 < P < 0.05.
n=&7. **p < 0.01.
*Calculated from mean values. For data see Fig. 2A. n for Na+, 12; K’. 24
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