Vol. 86, Issue 2, 536-540, February 1999
Heat acclimation does not alter rat mesenteric artery response
to norepinephrine
R. C.
Looft-Wilson,
R. D.
Matthes, and
C. V.
Gisolfi
Department of Physiology and Biophysics, Bowen Science Building,
The University of Iowa, Iowa City, Iowa 52242
 |
ABSTRACT |
Previous studies
have shown that heat acclimation raises the temperature threshold for
heat-induced splanchnic vasoconstriction in the rat (W. Haddad and M. Horowitz. Thermal Balance in Health and Disease,
Advances in Pharmacological Sciences. Basel:
Birkhauser, 1994, p. 203-208; M. Shochina, W. Haddad, U. Meiri,
and M. Horo-witz. J. Therm. Biol.
21: 289-295, 1996). We tested the hypothesis that heat acclimation
alters splanchnic resistance artery sensitivity to norepinephrine (NE).
Male Sprague-Dawley rats (n = 5) were acclimated to 35°C ambient temperature for 5-8 wk. Control
rats (n = 5) were maintained at
22-23°C ambient temperature for 5-7 wk. Small mesenteric
artery segments (2- to 3-mm length, 100- to 340-µm ID) were isolated,
cannulated at both ends, and pressurized to 50 mmHg. Artery luminal
diameter was measured in response to cumulative doses of NE
(10
9 to
105 M) by using video
microscopy. NE dose response was measured at 37 and 43°C bath
temperatures. There were no differences in constriction responses to NE
between acclimated and control rat arteries at either 37 or 43°C.
We conclude that acclimation does not alter rat mesenteric artery
sensitivity to NE.
isolated vessels; acclimation; rat
| |
INTRODUCTION |
EXPOSURE TO ACUTE HEAT STRESS results in splanchnic and
renal vasoconstriction (19). This vasoconstriction allows for greater diversion of blood flow to vascular beds involved in heat dissipation, such as the cutaneous vascular bed in humans. In the rat, recent studies have suggested that long-term heat acclimation (
14 days) raises the temperature threshold for heat-induced splanchnic
vasoconstriction compared with controls but increases maximal
vasoconstriction (5, 25). An increase in temperature threshold could
promote greater splanchnic blood flow during moderate heat stress,
reducing the possibility of ischemia-reperfusion injury and
increasing survival (9).
It is not clear whether the increased threshold is due primarily to
reduced splanchnic sympathetic output and therefore reduced local
norepinephrine concentration or to reduced vascular responsiveness to
norepinephrine (NE). A change in number and/or sensitivity of
adrenergic receptors after heat acclimation has been suggested by in
vitro studies of portal vein and aortic ring isometric tension (5, 25).
The purpose of this study was to test the hypothesis that heat
acclimation decreases the sensitivity of mesenteric resistance arteries
to NE. Small mesenteric arteries (<500 µm ID) confer significant
resistance in this vascular bed (14), and sympathetically derived NE is
the primary mechanism for increased splanchnic vascular resistance
during heat stress (20). Decreases in small mesenteric artery
sensitivity to NE could, therefore, be a possible mechanism for the
increased core temperature threshold for heat-induced splanchnic
vasoconstriction after heat acclimation.
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METHODS |
Animal protocol.
The experimental protocol was approved by the University of Iowa Animal
Care and Use Committee. Ten male Sprague-Dawley rats (250-300 g
body wt) were familiarized with a motorized treadmill by walking at 10 m/min, 0% grade, for 15 min/day for 6-7 days at 23°C. A
heat-tolerance test was then performed the day after the end of the
familiarization period. This involved exposing rats to 40°C ambient
temperature while they walked on a treadmill (12 m/min, 0% grade)
until their rectal temperature reached 41.5°C. We measured the time
required to elicit a rectal temperature of 41.5°C (heat-tolerance
time) and the area under the curve of the plot of rectal temperature
vs. time (thermal load).
Five rats were then acclimated to heat for 5-8 wk by exposure to
35°C ambient temperature for 12 h/day during the 12-h light cycle
(6:00 AM to 6:00 PM). They also performed treadmill walking (12 m/min,
0% grade) once per day at 35-40°C ambient temperature until
their core temperature reached 41°C, which took between 23 and 48 min. Core temperature was measured by a rectal thermistor (model 402, Yellow Springs Instruments, Yellow Springs, OH) and telethermometer
(model 44TA, Yellow Springs Instruments). This low exercise intensity
(12 m/min, 0% grade) was utilized to facilitate the rise in core
temperature, and it is unlikely to produce a training effect in rats
because much faster treadmill speeds are typically utilized to produce
a measurable exercise training effect (2). If the rat either lost
weight or refused to walk, a day of rest was given. Five control rats
were housed at 22-23°C ambient temperature for 5-7 wk.
Another heat-tolerance test was performed after the acclimation period.
Acclimation was confirmed by a greater heat-tolerance time and thermal
load after acclimation compared with controls.
Isolated vessel protocol.
Mesenteric artery segments were isolated from heat-acclimated rats 24 h
after the heat-tolerance test and from control rats 6-12 days
after the heat-tolerance test. Only 24 h of recovery were given to the
heat-acclimated rats because the heat-tolerance test resulted in a rise
in core temperature only 0.5°C greater than that during their daily
exposure while walking on the treadmill and did not represent a
significant heat stress for this group. The controls, however, were
given 6-12 days of recovery before isolation of arteries because
the heat-tolerance test would be a significant stress for this group.
To isolate mesenteric arteries, rats were anesthetized with
pentobarbital sodium (50 mg/kg body wt ip) and a midline incision was
made. The small intestine and associated mesentery were isolated with
silk sutures and removed. The mesentery was placed in a dissecting dish
with cold physiological salt solution [PSS containing (in mM):
119 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 25 NaHCO3, 1.2 KH2PO4,
and 5.5 dextrose, pH 7.4]. An artery segment (2- to 3-mm length,
100- to 341-µm ID) was removed and stripped of adhering connective
tissue by using a dissecting microscope (Olympus, Lake Success, NY) and
fine-tipped forceps (no. 5, Dumont Medical, Fine Science Tools,
Belmont, CA). The isolated artery segment was placed in a microvessel
chamber filled with PSS (15 ml), and one end was cannulated with a
glass pipette, secured with nylon opthalmic sutures (10-0), and flushed with PSS to remove any blood. The other end was then cannulated and secured.
The microvessel chamber was mounted on a microscope stage (inverted
microscope, model CK2, Olympus) and visualized by using a video camera
(charge-coupled device, Hitachi Denshi). The vessel was
superfused with aerated (95%
O2-5%
CO2) PSS maintained at 37°C
by circulating PSS (100 ml in total system: microvessel chamber, tubing, external reservoir) through a glass coil heated by an external
water bath (Masterflex HS console drive, Haake water bath, Cole-Parmer
Instrument, Chicago, IL). Vessel bath temperature was monitored by
using a digital thermometer with a copper-wire thermocouple (Omega
Engineering, Stamford, CT) placed within 1 cm of the vessel. The artery
was pressurized (no flow) and maintained at 50 mmHg by using a pressure
servo control system (Living Systems Instrumentation, Burlington, VT).
After pressurization, the distance between the cannulas was increased
to remove the buckling in the artery caused by pressure-induced
lengthening. Artery diameter was measured on screen by using a video
measurement system (model VIA-100, Boeckeler Instruments, Tucson, AZ).
Cannulated arteries were equilibrated at 37°C for at least 30 min.
Artery luminal diameter was then measured during cumulative addition of
NE
(109-105
M). Two minutes were allowed after each dose for the vessel response to
stabilize. The dose-response curve required ~24 min to complete. Doses above 106 M routinely
elicited vasomotion. Both maximal and minimal luminal diameters were
recorded during vasomotion, and the average diameters are reported.
Arteries were considered viable if constriction to NE
(105 M) resulted in >50%
reduction in luminal diameter and if subsequent addition of ACh
(105 M) resulted in
relaxation to >80% resting diameter.
After the NE dose-response and ACh response measurements were made at
37°C, the chamber was flushed with 400 ml of PSS, and the bath
temperature was increased and equilibrated at 43°C for 30 min. All
vessels returned to baseline diameter during equilibration. A second NE
dose response and ACh response were measured at 43°C. This 43°C
bath temperature was chosen because it is the highest reported core
temperature reached during heat stress after which some animals still
survive. Specifically, Fruth and Gisolfi (2) showed that several rats
survived 24 h after a heat-tolerance test in which core temperatures
reached 42.6-43.0°C. Ryan and Gisolfi (22) also showed that
responsiveness of isolated mesenteric arteries to NE was not
significantly different at 37, 42, or 43°C bath temperature.
Statistics.
Responses to the heat-tolerance test were compared in each group before
and after the acclimation period and between groups by using one-way
ANOVA. Responses to each dose of NE were compared between the control
and heat-acclimated vessels at 37 and at 43°C by using a two-way
ANOVA with repeated measures. The responses of each group were also
compared between temperatures by using a two-way ANOVA with repeated
measures. NE concentrations required to elicit threshold or 10%
(EC10), half-maximal
(EC50), and maximal (EC100) vasoconstriction were
calculated by using a computer program (Kaleidograph, version 3.0.2, Abelbeck Software) with concentration-response data fitted to the
equation Y = M1[1
e(M2×M0)],
where Y is the response obtained with
a given NE concentration; M1 is the maximal attainable response; M0 is
the concentration required for 10, 50, or 100% maximum contraction;
and M2 is a constant. EC10,
EC50, and
EC100 were compared between groups
by two-tailed unpaired t-test.
Significance in all analyses was determined by
P < 0.05.
| |
RESULTS |
Preexperimental heat-tolerance times and thermal loads were similar in
the heat-acclimated group [26.0 ± 1.87 (SE) min, 11.5 ± 0.9°C · min, respectively] and the control
group (18.2 ± 2.52 min, 9.5 ± 1.3°C · min, respectively). After
the experimental treatment, heat-acclimated rats had significantly
longer heat-tolerance times (44.9 ± 4.1 min) compared with controls
(26.4 ± 2.1 min) and compared with their preexperimental
heat-tolerance times. They also tolerated significantly greater thermal
loads (17.6 ± 2.7°C · min) compared with
controls (10.6 ± 3.5°C · min) and compared
with their preexperimental loads. There was no significant difference
in heat-tolerance times or thermal loads in control rats before and
after the experimental treatment. These results indicated that the
heat-treated rats were heat acclimated.
There was no significant difference in responses to NE at any dose
between arteries from control and heat-acclimated rats at either 37 or
43°C (Figs. 1 and
2). There was also no significant difference in responses of either arteries from control rats or arteries from heat-acclimated rats between temperatures (Figs. 1 and
2). Additionally, NE concentrations required to elicit
EC10, EC50, and
EC100 were not different between
arteries from heat-acclimated rats or arteries from control rats at
either temperature (Table 1). Vasodilation of
preconstricted arteries to ACh was also similar in all conditions
[control at 37°C: 94 ± 1.6 (SE)%; heat-acclimated at
37°C: 91.2 ± 1.9%; control at 43°C: 93.2 ± 0.9%;
heat-acclimated at 43°C: 87.2 ± 4.1%].
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Fig. 1.
Change in luminal diameter in response to a cumulative dose of
norepinephrine at bath temperature of 37°C. Zero on
y-axis represents baseline diameter
after equilibration in physiological salt solution. Luminal diameter
change of 100% would indicate total artery occlusion. Values
are means ± SE; n = 5 arteries
(from 5 different rats) in each group.
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Fig. 2.
Change in luminal diameter in response to a cumulative dose of
norepinephrine at bath temperature of 43°C. Zero on
y-axis represents baseline diameter
after equilibration in physiological salt solution. Luminal diameter
change of 100% would indicate total artery occlusion. Values
are means ± SE; n = 5 arteries
(from 5 different rats) in each group.
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Table 1.
Effective concentrations of norepinephrine to elicit
EC10, EC50, and EC100
vasoconstriction at 37 and 43°C bath temperatures in control and
heat-acclimated rats
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DISCUSSION |
These data do not support the hypothesis that heat acclimation
decreases the sensitivity of mesenteric arteries to NE. These data
provide evidence that heat acclimation does not change sensitivity or
the maximal vasoconstriction response of rat mesenteric arteries to NE
either at basal temperature (37°C) or during simulated heat stress
(43°C). Other investigators have suggested that splanchnic blood
flow is increased after heat acclimation (5, 9, 25). One mechanism that
could explain this increase in splanchnic blood flow after heat
acclimation would be a reduction in responsiveness to vasoconstrictor
agents. To test this possibility, we examined the responses of isolated
mesenteric small arteries to NE. NE is the expected neurotransmitter
responsible for mesenteric vasoconstriction during heat stress because
the mesentery is heavily innervated by sympathetic-adrenergic fibers
(4) and splanchnic sympathetic output increases during heat stress in
the rat (3) and other species (20). Because our data indicate no change
in sensitivity or maximal response to NE, this suggests that the
greater splanchnic blood flow observed by other investigators (5, 9,
25) may be due to altered splanchnic sympathetic output or
concentrations of humoral vasoactive agents during heat stress. These
possibilities have not yet been examined; however, the increase in
plasma NE and epinephrine concentrations during exercise in the heat is attenuated in humans after acclimation (16). This reduction in plasma
catecholamine concentration could be the result of a decrease in
sympathetic output to the splanchnic vasculature after acclimation.
Increased basal splanchnic blood flow after heat acclimation was
initially observed by Horowitz and Samueloff (9), who showed that
splanchnic organ and liver blood flow was greater after 2 wk of heat
acclimation. These experiments used radiolabeled microspheres to
measure blood flow. It was also recently reported that portal vein
blood flow during heat stress is elevated after heat acclimation.
Specifically, portal vein blood flow remained constant at near-basal
levels in control rats until a core temperature of 41°C was
achieved, after which it declined. In heat-acclimated rats, portal vein
blood flow tended to rise until a core temperature of 41°C was
achieved and then precipitously fell at 42°C to a greater extent
than in controls (5, 25). The authors of these latter studies suggested
that heat acclimation (14-30 days) increased the core temperature
threshold for splanchnic vasoconstriction and produced a greater
maximal vasoconstriction. This maintenance of portal blood
flow during increases in core temperature below 41°C, particularly
in control rats, is in conflict with other existing data. Kregel et al.
(11) found that, during heat stress in the rat, blood flow through the
superior mesenteric artery decreased linearly with increasing core
temperature from 38 to 41.5°C. A similar reduction in splanchnic
blood flow with increasing core temperature was also observed in humans
(20). One might expect similar blood flow changes through the portal
vein during heat stress. This is because blood flow through vascular
beds supplied by the superior mesenteric artery, along with the
inferior mesenteric artery and coeliac artery, constitutes the blood
supply to the portal vein (18). It is not clear why such differences were found in portal vein blood flow (5, 25) and superior mesenteric
artery blood flow (11) during heat stress in these studies, given that
similar blood flow measurement techniques were used. On the basis of
the present data, if portal blood flow, and in turn splanchnic blood
flow, is greater during heat stress after heat acclimation compared
with that of controls, it is more likely due to decreased sympathetic
or humoral stimulation rather than to decreased sensitivity of the
vasculature to NE.
With heat acclimation in humans, the onset of sweating occurs at a
lower core temperature threshold and there is a greater rate of
sweating at a given core temperature (15, 21). In rats, there is
greater saliva production (the primary mechanism for evaporative
cooling in the rat) that occurs at a lower core temperature threshold
(6). Cutaneous vasodilation also occurs at a lower core temperature in
humans (2, 17) so that skin blood flow is greater at a given core
temperature. In rats, however, the core temperature threshold for tail
skin vasodilation is increased after heat acclimation (23, 24). This
attenuation of the increase in skin blood flow during heat stress would
presumably allow reduced splanchnic vasoconstriction, and thus
increased splanchnic blood flow, at a given core temperature and still
maintain circulatory stability (19).
Previous studies have suggested that after heat acclimation there are
changes in effector organ sensitivity to catecholamines. For example,
in the rat salivary gland (the primary evaporative cooling organ in the
rat), 30 days of heat acclimation resulted in increased NE sensitivity
due to increased muscarinic acetylcholine-receptor density (5). It is
also well known that the increase in heart rate with heat stress is
attenuated after heat acclimation in both humans (21) and rats (8),
which is concomitant with a greater stroke volume. Several studies (10,
12, 13) found that the greater stroke volume was due in part to
increased contractility and functional mechanics of the heart after
heat acclimation in the rat. Moreover, after heat acclimation, rat
aortic rings develop greater isometric force to phenylephrine and
vasodilate less to isoprenaline (25); however, the significance of this
observation is unknown. There is also evidence that rat portal vein
vasoconstriction to 
-adrenergic-receptor stimulation and tail artery
vasoconstriction to NE increase after heat acclimation
(5). These data provide ample evidence that some end-organ
responses are altered after heat acclimation. Similar types of
alterations in the mesenteric arterial vasculature, however, are not
supported by the results of the present study. Adrenergic receptor
populations (1,
2, 
2) in these mesenteric
vessels may be altered with heat acclimation; however, no change in the
functional response to NE was observed in this study.
In summary, the rat mesenteric small-artery sensitivity and maximal
vasconstriction to NE are not altered by 5-8 wk of heat acclimation. This indicates that the proposed increase in splanchnic blood flow during heat stress after heat acclimation, compared with
controls, is not due to alterations in mesenteric vascular responsiveness to sympathetic output.
| |
ACKNOWLEDGEMENTS |
The authors thank Ting Xia for performing the heat-acclimation
protocol and the heat-tolerance tests and Joan Seye for assistance in
manuscript preparation.
| |
FOOTNOTES |
This work was supported by National Institute of Arthritis and
Musculoskeletal and Skin Diseases Grant AR-40771-05A1.
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. §1734 solely to indicate this fact.
Address for reprint requests: C. V. Gisolfi, Dept. of Physiology and
Biophysics, Bowen Science Bldg., The Univ. of Iowa, Iowa City, IA
52242.
Received 16 June 1998; accepted in final form 8 October 1998.
| |
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Abstract
Introduction
