Effects of Hypothermia on Norepinephrine
Release and Effector Response in
Isolated Perfused Cat Spleen
By Ake Wennmolm, Per Hedqvist, and Lennart Stjdrne
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ABSTRACT
The effects of hypothermia on the sympathetic neuroeffector junction were
studied in the isolated perfused cat spleen. The experiments were aimed at
determining the temperature dependence both of prejunctional events, i.e.,
efflux of norepinephrine from the sympathetic nerves in response to electrical
stimulation or to indirectly acting sympathomimetic amines like tyramine or
phenethylamine, and of postjunctional events, i.e., the contractile response of
the smooth muscle to the norepinephrine liberated. Hypothermia was found to
depress both the norepinephrine efflux in response to stimulation and the
contractile response to the endogenous norepinephrine released, as well as to
exogenous norepinephrine. However, these processes were found to respond
differently to temperature change. Thus hypothermia depressed norepinephrine
release as well as the pressor responses to tyramine and phenethylamine much
more strongly than the corresponding responses to electrical nerve stimulation
or the pressor response to exogenous norepinephrine. The results strongly
support the concept that sympathomimetic amines release norepinephrine from
sympathetic nerves by mechanisms which differ from those mediating
norepinephrine release induced by depolarization.
ADDITIONAL KEY WORDS
tyramine
temperature
• The sympathetic nervous system plays an
important role in thermal homeostasis
during general exposure to cold, which induces
increased activity in the sympathetic nerves,
as reflected by a marked increase in the
urinary excretion of norepinephrine (NE)
(1). However, less is known concerning the
direct local effect of hypothermia on sympathetic nerve function, such as processes
involved in conduction of impulses in C fibers,
neurotransmitter release, and sensitivity of the
smooth muscle to the transmitter released.
Nerve conduction is known to be temperature dependent. Thus, Franz and Iggo (2)
found that in nonmyelinated nerve fibers there
From the Department of Physiology, Karolinska
Institutet, S 104 01 Stockholm 60, Sweden.
This investigation was supported by grants from
Stiftelsen Lars Hiertas Minne and from the Swedish
Medical Research Council, Project B 70-14X-97-06B
and Project B 70-14X-2705-02.
Received February 11, 1970. Accepted for publication May 13, 1970.
CircnUtion Rtsurcb,
Vol. XXVII, July 1970
sympathetic nerves
nerve stimulation
smooth muscle
was a progressive reduction in conduction
velocity as the temperature was lowered from
37° down to 8°C, at 10°C being reduced to 15
to 20$ of the 37°C value.
The effect of hypothermia on neurotransmitter release has so far not been extensively
investigated. In studies of the release of NE
from the sympathetic nerves of the isolated
cat spleen Kirpekar et al. (3) found that
reduction of the perfusion temperature to
15°C markedly reduced the amount of NE
released in response to nerve stimulation or to
infusion of potassium. However, the authors
did not present quantitative data concerning
the extent of reduction of NE release during
hypothermia.
The spontaneous release rate of NE from
isolated bovine splenic nerve granules was
found to be reduced to about 10 percent when
the temperature of the incubation medium
was reduced from 37° to 20°C (4, 5).
However, the significance of the spontaneous
release of NE from isolated granules with
19
40
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respect to the release of NE from the nerves in
response to depolarization is not known.
The sensitivity of smooth muscle to catecholamines has been reported to be altered by
low temperature. Thus Smith (6) stated that
vessels stimulated by epinephrine contracted
and relaxed more slowly at 17° than at 37°C.
Keatinge (7) found that the response of
isolated vascular strips to epinephrine progressively decreased when the temperature was
lowered from 36°C, being completely abolished between 12.5° and 10°C. In using a
similar preparation, Sams and Winkelmann
found a decrease in catecholamine-induced
contraction and in addition a changed response pattern when the temperature was
lowered (8). On the other hand, hypothermia
has been reported to potentiate certain
smooth-muscle effects of catecholamines.
Thus, the cutaneous veins of dogs contract
more strongly in response to NE when the
temperature is lowered from 37° down to
17°C (9). In addition, epinephrine has been
shown to inhibit the spontaneous activity of
the isolated rabbit small intestine more
effectively at 30° than at 38°C (10). An
increased response during hypothermia has
also been found in the isolated guinea pig vas
deferens by Delia Bella et al. (11). They
found that the contraction on nerve stimulation increased 5 to 10 times when the
temperature was lowered from 32° to 20°C.
Methods
Cats of both sexes weighing 2.5 to 4.5 kg were
used for the study. The experimental animals
were anesthetized with sodium pentobarbital
(30 mg/kg ip) and heparine (1000 IU/kg iv).
The abdomen was opened by a midline incision
and the spleen together with its vascular and
nervous supply was isolated. The organ was
transferred to a perfusion chamber and perfused
with a physiological salt solution (in MM: NaCl,
103.4; KC1, 4.8; MgSO, • 7 H O , 1.2; CaCl2, 2.5;
Na-pyruvate, 4.8; Na-1-glutamate 2.2; KH2PO4,
1.0; Na HCO3> 25; dextrose, 11.5; ascorbic acid,
0.11) aerated with 5% CO2 in O2. The flow rate
was kept at 7.5 ml/min by a constant rate
perfusion pump. Perfusion pressure was registered with a Statham pressure transducer connected to a Grass Model 7 polygraph. The
effluent from the spleen was divided into 10 ml
WENNMALM, HEDQVIST, STJARNE
portions by means of an automatic fraction
collector. Supramaximal electrical stimulation of
the splenic nerves was carried out with platinum
electrodes, using a Grass S4 stimulator, operated
at a frequency of 10 cps, a duration of 2 msec,
and a stimulation strength of 10 to 15 volts, for
stimulation periods of 20 seconds, separated by
an interval of 10 minutes. The temperature was
measured in the perfusing medium, in the
perfusion chamber, and on the surface of the
spleen.
The experiment was started by an intra-arterial
infusion of 30^ic of tritium-labeled dl-NE
(specific activity 5 mc/ynmole, New England
Nuclear Corp.). After washing for 30 minutes by
perfusion with NE-free Rrebs-Henseleit's solution,
the spleen was exposed either to nerve stimulation
or to intra-arterial injection of NE (0.3 to 0.4
/j.g), phenethylamine (20 ug) or tyramine (20
/i.g). The temperature of the system was varied
stepwise between 8° and 37°C, both increasing
and decreasing the temperature. The nerve
stimulation or injection of NE, phenethylamine,
or tyramine was carried out at each step.
The time course and profile of the NE output
from the sympathetic nerves in the cat spleen was
monitored by measuring the amount of radioactivity in each 10-ml fraction of the effluent from
the perfused organ. The radioactivity of the
different samples was determined by counting
0.5-ml aliquots in a Packard Liquid Spectrometer
using a 7:3 toluene-absolute ethanol solution
containing 4 g of 2-5-diphenyloxazole and 100
mg of l-4-bis-2 (4-methyl-5-phenyIoxazoIyI) benzene per liter of toluene. Quenching was
monitored by internal standards of 3H-dl-NE.
Results
According to previous evidence, almost all
of the radioactive material released in response to nerve stimulation or to injection of
tyramine in the preparation used consists of
intact NE (12). Reduction of the temperature
of the spleen depressed the efflux of NE and
the pressor effects caused by electrical nerve
stimulation as well as by phenethylamine or
tyramine (Figs. 1, 2). However, the tyramineinduced overflow of NE was clearly more
temperature dependent (Fig. 2). Similar
results were obtained with phenethylamine.
The relative fall in the NE-releasing effect of
tyramine was significantly more marked than
that of electrical nerve stimulation, both at
25° and 15°C.
The average resting perfusion pressure was
CircMlttion Ruurtb,
Vol. XXVII, July 1970
41
SYMPATHETIC NERVE FUNCTION IN HYPOTHERMIA
I*
mm Hg
cr >
o ir
in z
65
UJ
? o
cpm
10 000
I
5.000
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t
t
10
t
T
25
10
25
37
TEMP °C
FIGURE 1
Typical experiment showing the effect of hypothermia on outflow of radioactivity and pressor
response to 20 seconds of nerve stimulation in the isolated cat spleen.
% of
UJ
£ 2 control
o o 100
Q.
I/)
UJ
Q:
r
uJ
->
Z
v
—
z <
o <
q
50
>
0L
35
30
25
20
15
10
TEMP.°C
FIGURE 2
Effect of hypothermia on the outflow of radioactivity from isolated cat spleens in response
to nerve stimulation or tyramine injections. Vertical bars = means ± SE (n = 5) in percent of
control values obtained at 37°C. Nerve stimulation and tyramine injection values statistically
separated at 25°C (P < 0.01) and at 15°C (P < 0.02). TA = tyramine.
about 25 mm Hg. During hypothermia this
pressure rose to 30 mm Hg in some of the
Oremltiicm R,lurch,
Vol. XXVII, J*I) 1970
experiments. At 37°C the pressor response to
nerve stimulation was 20 to 30 mm Hg. This
WENNMALM, HEDQVIST, STJARNE
42
30
constant amounts of NE (Fig. 4). The pressor
responses to tyramine and phenethylamine
were about 5 mm Hg at 37°C in all
experiments. This response rapidly decreased
when the temperature was lowered, and was
completely lost at around 25°C.
20
Discussion
mm Hg
z
o
a.
hi
z
z
o
o.
I/)
UJ
10
a
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
35
30
25
20
TEMP. 'C
15
30
FIGURE 3
Regression line showing the effect of graded hypothermia on the pressor res^Jonse to nerve stimulation
in three isolated cat spleens.
mm Hg
75 r
Id
z
o
t—
HI
z
o
a.
)
25
o
UJ
a.
35
30
25
20
TEMP.'C
15
10
FIGURE 4
Regression line showing the effect of graded hypothermia on the pressor response to injection of NE
in three isolated cat spleens.
response progressively decreased with the fall
in temperature (Fig. 3) to be almost completely abolished at temperatures below 15°C.
Virtually the same effect was obtained when
nerve stimulation was replaced by injection of
Hypothermia was found to depress the NE
efflux and the pressor responses both to
depolarization and to tyramine or phenethylamine, although to a different extent. The
reduced NE overflow on nerve stimulation
might be due to direct effects of hypothermia
on nerve conduction, on release of NE from
the nerves, or on removal or enzymatic
inactivation of the NE released. Nerve conduction is blocked between 1° and 7.1°C (2,
13). Thus, impaired nerve conduction might
have influenced the overflow of NE to some
extent in the lower temperature range of the
present study. However, at moderate hypothermia impaired conduction was probably
not of any major importance.
Release of NE from the nerves in response
to nerve stimulation has previously been
shown to be depressed by hypothermia (3).
This is in accordance with the present results.
The process of spontaneous release of NE
from isolated bovine splenic nerve granules is
highly temperature dependent. The basic
release rate at 37° is 8 to 10 times higher than
that at 20°C (4, 5). However, the outflow of
NE in response to nerve stimulation in the
present series was far less temperature dependent, the ratio of NE efflux at 37° and that at
20°C being about 1.5. Thus it seems unlikely
that the release of NE from the nerves in
response to depolarization is immediately
dependent on the same mechanisms as those
determining spontaneous release of NE from
isolated nerve granules in the temperature
range mentioned. Reduction of temperature
below 20°C produced a pronounced depression of the overflow of NE in response to
nerve stimulation. During such extreme hypothermia the temperature dependence of the
outflow response to nerve stimulation was
close to that found for tyramine and phenethyCircuUtion Rtuircb,
Vol. XXVII, July 1970
SYMPATHETIC NERVE FUNCTION IN HYPOTHERMIA
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lamine in the 37° to 25°C temperature
range.
On the other hand, the temperature dependence of the phenethylamine- or tyramineinduced release of NE from the nerves was
more closely related to that of spontaneous
release of NE from isolated nerve granules.
This may be of particular significance since
the NE-releasing effect of tyramine on the
nerves appears to be exerted in part by direct
action on the granules (14). At this level
tyramine may act both by displacement of
granule-bound NE (15) and by inhibition of
reuptake into the granules of the NE released,
spontaneously or by tyramine. Thus the
observed similarity in temperature dependence between spontaneous release of NE
from isolated granules in vitro and tyramineinduced release of NE from the whole neuron,
may indicate that spontaneous release of NE
from granules in situ may be rate limiting for
the effect of tyramine on the nerves. Hypothermia may in addition interfere with the
effects of tyramine at other levels, such as on
the uptake of tyramine into the nerves (12)
and on the tyramine-induced inhibition of
reuptake into nerves of the NE released
(16,17).
The vasoconstrictor effect of adrenaline has
been shown to be reduced by low temperature
(6, 7). However, it has recently been shown
that the response to NE is markedly increased
in dog's cutaneous veins (9). In the present
study the pressor effect in response to nerve
stimulation gradually decreased when the
temperature was lowered. Not only was the
amplitude of the response altered, but also the
pattern of the curve. At 37°C there was a
sharp initial rise at the beginning of the nerve
stimulation or NE injection, but during
hypothermia this sharp rise became gradually
less pronounced, giving the curve a more
flattened profile.
In conclusion, hypothermia depresses several functions of the sympathetic neuroeffector
system of the isolated perfused cat spleen,
apparently by reducing NE release from the
nerves as well as the sensitivity of the smooth
muscle to NE. However, there is a marked
Circulation Ruurcb,
Vol. XXVll, J*LJ 1970
43
difference in the influence of hypothermia on
the effect of the two stimuli used for inducing
NE release, in the 37° to 20°C temperature
range. Thus, the "physiological" release of NE
induced by nerve impulses is much more
resistant to hypothermia than the "pharmacological" release induced by sympathomimetic amines. This supports the concept that
these two types of NE release depend on
distinctly different mechanisms. It also indicates that "physiological" release, i.e., secretion on depolarization, is not immediately
dependent on spontaneous release of NE from
nerve granules, while "pharmacological" release may well be so (cf. 18).
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CircmLaio* Ruurcb,
Vol. XXVII, July 1970
Effects of Hypothermia on Norepinephrine Release and Effector Response in Isolated
Perfused Cat Spleen
ÅKE WENNMALM, PER HEDQVIST and LENNART STJÄRNE
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Circ Res. 1970;27:39-44
doi: 10.1161/01.RES.27.1.39
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