Life Bcienoes Yol . 5, pp . 485-493, 1966 " Printed in Great Britain.
Pergamoa Press Ltd.
SFFECT OF ACUTE COIa SI~OSURE OF AATM6TR pN THE PH(78PHORYIASS ACTIYITI IN HEART Naraajan S . Dhalla Department of Pharmacology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania Department of Pharmacology, School of Medicine, University of Pittsburgh* Pittsburgh, Pennsylvania
(Received 28 October 1965 ; in final ft~rm 11 January 1966) Shivering in response to a cold stimulus was impaired in adrenal demedulfated animals with peripheral sympathetic blockade ; however, if the .adrenal medulla was functional, shivering was possible even in the presence of peripheral sympathetic blockade .l
Besides adrenal medulla, the sympathetic aerwus
system is considered to play an essential role for providing endogenous substrates in order to meet the high metabolic requirement of gency eonditions such as cold exposure . l'2
This conclusion is based on the
fact that chemically sympathectomized-demedullated rata when exposed to a cold stimulus were unable to mobilize free fatty acids and glucose.
in adrenal demedullated rats, the cold exposure was able to readily mobilize free fatty acids but the output of glucose was insufficient to raise the plasma levels .2
The cold exposure has also been shown to reduce muscle and
liver glycogen contents .3s4 Recently, Berti et a15 have observed s marked fall in body temperature and heart rate associated with changes in cardiac electrical eves is immuaosympathectamized-medullectamized rats on keeping them in a cold roam at for 2 hr .
On the other hand, i.imnm~R,ympathectomyr or medullectomr alone failed
to produce such alterations in animals exposed to cold .,
The stimulation of
the sympathetic nerwus system6 has been demonstrated to activate cardiac phosphorylsse which is known to participate mainly in the breakdown of glycogen . * Present address 485
HEÀRT PHOSPHORYL~3E ACTIVITY
Yol . 5, No . 6
It was therefore though of interest to investigate the influence of cold exposure on the phosphorylase activity in the heart .
The effect of~cold in rats
and rabbits pretreated with various agents known to interfere with sympathetic nerwus system through different menhanisma was also studied . Materials and Methods Male albino rats study.
(225-250 g) and. rabbits (1 .8-2 .0 kg) were used in this
The rats were kept in a cold roam at 4°C for 1 to
for 2 hr before sacrifice.
hr ; while rabbits
In one group of rata, bilateral adrenalectomy or
adrenal demedullation was performed 5 days prior to the experiment and ahem operated rats served as control.
These operated rata were given l~, saline in-
stead of tap water for drinking and all the animals were fed ad lib . with Purina. Chow diet .
The roam temperature was about 25°C.
Drugs dissolved in
normal saline were injected intraperitoneally and control groups of animals received saline .
Nethalide hydrochloride was given to rats in two doses of
mg~kg and 2 mg~kg within 1~ hr and the animals were sacrificed
the second injection .
The first injection,
(10 mg~kg) each of bretylium
tosyl.ate and guanethidine sulfate, was followed by another injection of these drugs (5 mg~kg each) 20 hr later; i .e . 2 hr before the rabbits were killed . Both rats and rabbits received reserpine in two injections (2 .5 mg~kg each) at 0 and 24 hr and the animals were killed 48 hr after the first dose . animals were sacrificeu by a blow on the head .
In one set of experimeata~
rats were anesthetized with pentobarbital sodium (50 mg~kg) 15 min before sacrifice and an equal volume of saline was injected into control rats .
hearts were quickly removed and kept in empty beakers already placed in an ice bath .
The phosphorylase activity was determined as described earlier.?
total phosphorylase contents did not change under sny of the experimental conditions, the phosphorylsse a activity was expressed as ~, of the total enzyme .
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~AET PHOSPSO$YLAßE ACTIVITY mear~z 1
Effect of exposure of rats to cold on the phosp~horylase activity in atria sad ventricles . Rats were kept in a cold room at 4°C for different time intervals . After removal of the heart, whole atria sad ventricle apex (about 100 mg) were dissected out imme diately for enzyme determination. '~" denotes the number of " an;+hala used far each experiment . "P" and '~ refer to the level of signifil cance as oampared with each control valve Pram rats without cold exposure in their respective group.
Procedure for animal sacrifice A.
Blow on the head
50 ~s/~ (15 min before
Phosphorylase a activity (~ of total) Mean + 9 .E .
71 .4 _+ 1.90
65 .6 _+ 2 .61
80 .6 + 2 .11 P<0.025
87 .5 + 2 .33
84 .2 + 2 .27
86 .4 + 3 .20
54 .2 + 3 .74 -
68 .6 + _ 3.17
69 .4 + 2 .09 Pl<0.05
78 .8 + 2 .08 Pl<0.05
Results Exposure of g "+imalR to cold for 1 to
hr was observed to significantly
increase the phosphorylase a activity in both atria and ventricle of rate sacrificed either by a blow on the head 0r pentobarbital anesthesia (Table 1) . It was found that pentobarbital injection in normal rats decreased enzyme activity in ventricle but failed to decrease phosphorylase e activity is atria as compared with control group of rats receiving a saline injection under similar conditions .
Since the atrisil and ventricular tissues responded to
cold exposure in a similar manner, it was decided to use either whole heart or ventricular tissue for further studies.
~d~tT PHCSPHO$YLAßE ACTIPITT
Yol . 5, Ao . 6
TABI~ 2 Effect of cold exposure oa cardiac phoephory]sée in adrenelectemized or drug treated rata . Rats vere kept is cold rocs! at 4oC for 2 hr and killed by a blow oa the head. The vhols heart vas removed for enzyme determinatia~n . Figures vithia parenthesis are the number of animals used for each experiment . ~" refers to the level of significance as ooorpared.t o control value in vertical co i+~+ while ~P " denotes the ].evel of significance oa cemparisoa between ani~ia exposed ~ cold and n.~ia not exposed to cold in each group. Phosphorylase é activity (~, oT total) Dtéaa + $,E. Animals not exposed to cold
Animals exposed to cold.
69 .5 + 2 .03 (16)
78 .8 + 2 .1A
67 .8 + 2 .46 (6) ~0 .05
71 .4 + 1.32 (S)
70 .4 + 1:04 (4) P>o,o5
72 .3 ± 0 .93 (5)
Rese ine 5 ms%/kB
56 .6 + 3.18 (8) P<0.01
68 .8 _+ 1 .44 (8)
1Pethalide 5 ms/ka
53 .8 + 1.84 (7) P
54 .9 _+ 1.76 (8)
Table 2 aho~s the effect of cold exposure is adrenalectamized or adrenal demedullated rats and lade .
pretreated with either reserpine or with aetha-
$rote sham operated anima~a did notshow any significant (P)0 .05) dif-
Terence in the heart phoaphorylsae a activity from the control, the values were grouped -together .
Adrenalectemy as demedullation did not decrease the
phosphorylase a activity is hearts Pram rata unexposed to cold .
cold exposure failed to elevate phoephory]see a levels is seams Tram adrenalectemized or demedullated rata .
Pretreatment of rata with reaerpine decreased
phosphory]sse a activity in hearts Tram cold. unexposed
but the cold
exposure oT reserpinized rats aigaiTicantly increased the enzyme activity . Oa the other hand, in nethalide treated rats, co]d exposure did not elevate
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HEAß'i' PS08PHCRYLABE ACTIVITY
TAHLE 3 Effect of cold exposure on cardiac phoaphorylase is control and drug treated rabbits. Rabbits were kept in cold roam at 4°C for 2 hr and killed by a b] .ow on the head . A portion of the right ventricle (about 200 mg) was dissected out for enzyme determination . Figures within parenthesis are the number of an "P" imals used in each study. refers to the level of sign_i.ficance as compared to control in vertical corn, while "P1" denotes the level of significeace on campariean between animals exposed to cold and anima],s not exposed to cold in each group . Phosphorylase e, activity (~, of total) Mean + S .E . Animals not exposéd to cold
Animals exposed to cold
49 .7 ± 3 .69 (u)
63 .5 _+ 2 .31 (6)
31 .9 + 2 .42 (8) P
50 .8 + 2 .40 (6)
33 .1 _+ 1.76
56 .4 _+ 2 .33
4b .2 _+ 3 .29 (7) P>o .o5
58 .6 + 1.54
the phosphorylase e activity in the heart, although like reserpine, nethalide reduced the enzyme activity in hearts Pram control animals at room temperature. It was considered of interest to find. if cardiac phosphorylase in rabbits exposed to cold responds like that in rats and also to study whether sympathetic blockade by bretylium and gusnethidine shows similar results as with reserpine.
When rabbits were kept in a cold room for
hr, the phosphorylase
a activity in myocardium was increased significantly (Table 3) .
of rabbits with either reserpine or guanethidine decreased the phosphox~ylase a activity in animals unexposed to cold, but the exposure of these rabbits to cold significantly elevated the enzyme activity .
Treatment of rabbits with
bretylium did not decrease phosphoxylase a levels in hearts from cold unexposed animals and the exposure of these animals to cold significantly elevated the enzyme activity in the myocardium .
HEART PHOSPHOxYL~ßE lCTIYITY
Yol . 5, Ro . 6
Mecusaion In control hearts from rats and rabbits unexposed to cold, the major portion of plwephorylase was observed to be in the active form . ment with earlier observations under similar conditione, 6 ~7e 8
This is in agreeand may be a
consequence of the sympathetic stimulation due to the procedure involved in animal sacrifice;6 or anoxia, maSy have played same role in causing high phospharylase a activity .9
Heart phoaphorylase in nor °i ~n .~~R was not affected
by adrenalectc~r but was decreased by the pa"ior mini stration of pentobarbital or reserpine.
These results support the fin s of Hess et a1 .6
observed that pentobarbital decreased phosphorylase a activity in ventricle but not in atrial tissue .
Such difference in the rat atrial and ventri
glycogenolysis, in response to acetylcholine and epinephrine, was also demonstrated by Vincent sad
Whether this mqy be a reflection of the
differences in the fuactioaal activities of these two areas of the heart, remains to be demonstrated . Aethe,lide, a potent ~- adrenergic receprtor blocking drugu end guaaethidine, en adrenergic neuron blocking agent producing cetecholemine depletion in heart like that of reeerpine,~ xere found tô lower the phoaphorylase a activity in hearts Pram normal animals .
These observations support the hy-
pothesis extended by Hess et a16 about the influence of sympathetic nervous system oa heart phosphorylase is Animals under normal conditions .
bretylium which is considered to b].ock the release of catecholaminea from sympathetic nerve endings in the tissue,
did not significantly decrease the
enzyme activity in rabbit heart and this is is contrast to the finding of Heea
~ ~.6 The present observation, that phoaphorylase a activity in the heart we,s increased by an acute cold stimulus to the animal, is not surprising in view of en increased metabolic requireme~ "for body funetion .l
However, it shoul3
be pointed out that the exact role of phoaphorylase in the heart is not cleea .
HEART PH08PSCRYL~$E ACTIVITY
Yol. 5, No . 6
bcposure of animals to cold has been reported to elevate pla®ta levels of l4,15,16 cstecho].aminea, corticosterone and thryoid hormones .
ga8rd and Hess l7 have documented various studies demonstrating augmentation of heart phosphorylase a activity by such hormones .
It seems therefore that
increase in phosphorylsse a activity in response to cold may be due to the interaction of catecholsmines and other hormonal agents secreted in the body under such a condition. Since the treatment of animals with nethalide abolished the phosphorylase activating effect of cold exposure, it is apparent that catecholsmines play a major role in eliciting such a metabolic action in the heart .
The increase in
heart phosphorylsse a activity is response to a cold stimulus seems to be indirectly mediated through the release of catecholsmines from thé adrenal medulla rather than fY~am the peripheral sympathetic nerve endings is tissues. This conclusion is based on the follaarlng observations :
(a) in adrena,lectam-
ized or adrenal demedul]ated rata, cold exposure failed to elevate cardiac enzyme activity ; (b) treatment of Ani,~wiR with agents such as bretylium, guanethidine and reserpine did not alter the ~osphorylase activating effect of cold exposure .
It should be pointed out that bretylium and guanethidine
have been found to have no effect oa adrenal medulls.~~ l9
pine has been shown to reduce the catecholamine contents of adrenal medulla, 2C the adrenal medu llary secretion in the reeerpinized preparations is considered to be adequate enough for the ftimctional requirement of the animal . 21 Si~ary Acute cold exposure of rats and rabbits at activity in the myocardium .
increased phosphorylase a
Ia adrenalectomized or adrenal demedullated rats,
the levels of cardiac phosphorylase a were not different from those in normal rats ; however, exposure of such animals to cold failed to elevate the en~rme activity in the heart .
Pretreatment of rats with nethalide decreased the
phosphorylase a activity as well as abolished the effect of rnld exposure .
HEART PHOSPH08YL~SE ACTMTY
Qol . 5, No. 6
When the rats and rabbits were treated with reserpine, the cardiac phosphorylass activity was decreased but the action of cold exposure was not altered . unlik e guanethidine, bretylium was unable to decrease the phosphorylase a activity in rabbit heart and the animals treated with these agents shouted an increase in enzyme activity in response to cold .
The results indicate that in
emergency conditions suçh as cold exposure, the heart phosphorylase is mainly influenced through the release of catecho]emines from adrenal medulla, . Acknowledgements - The author is gratefl~l. to Professor N . Haugaard and to Professor P. L. McLe,in for providing facilities for this work. References 1.
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