ACETYLCHOLINE AND ADRENALINE-NORADRENALINE
SENSITIVITY IN DENERVATED PAROTID GLAND OF MONKEY
By
J.
C. PAUL AND
J.
C. DAVJD.
Department of Pharmacology, Christian Medical College, Ve!lore
(Received March 31, 1958.)
The mechanism underlying a phenomenon of such a general occurrence
as the supersensitivity after denervation has often been attributed to a decrease
of the enzyme cholinesterase in the tissues concerned. Different organs exhibit
varying degrees of hypersensitivity; skeletal and smooth muscle, glands,
autonomic ganglion cells and neurones of the spinal cord and brain all develop this post-denervation sensitisation. Cannon (1939) in his 'Law of Denervation' states "When the efferent neurones in a unit are sectioned, an increased
iritability to chemical agents develops in the denervated segment".
It is the purpose of this investigation to study the secretory effect of
acetylcholine, adrenaline, nor-adrenaline and electrical stimulation on the
normal and denervated parotid gland. It is realised that even though
such experiments may offer some data, the methods of study available are
rough estimates and the significance of the results must be cautiously
judged.
The salivary glands being paired, offer a suitable and simple means of
studying supersensitivity after denervation, as one gland can serve as con trol
AURICUlO·
TEMPORAL
NERVE
~,,<)
<)~\<)
r}-
PUCCAl GltlPllO
\'~~
INFERIOR fACIAL VEIN
lYMPH NODE,
SUBMlXILll\RY GLAND
SUPERIOR FACIAL VEIN
EXT. JUGULAR VEIN
Fig.
438
DENERVATED PAROTID GLAND
to the other. The salivary glands receive both sympathetic and parasympathetic innervation. Stimulation of either elicits secretory activity of these
glands. Anatomical findings of Kuntz and Richins (1946) suggest that the
secretory tissue is innervated directly through the parasympathetic. In the
parotid gland of the monkey, the anatomical arrangement of the fibres
permits a study of the effects of postganglionic parasympathetic denervation
in this gland. The inferior salivatory nucleus provides preganglionic fibres
to the glossopharyngeal nerve. These synapse in the otic ganglion with
postganglionic neurones supplying the parotid gland (Kappers, Huber and
Crosby, 1931). (Fig. I.)
METHOD
Six adult monkeys of the species macaca radiata were used. The animals
were anaesthetised with Ether and Nembutal, and a one inch vertical incision was made. (Fig. 2). The right parotid gland was pushed medially and
the auriculotemporal nerve divided and portion of it was removed under
aseptic conditions. The wound was closed with continuous cotton suture.
All the animals were placed under identical conditions of colonisation for a
fortnight.
Fig. 2
A post-operative interval of a fortnight was allowed. The animals were
then anaesthetised, and the duct of the gland was dissected just before its
entry into the mouth and exteriorised. Standard polythene resilient tubing
was tied into place, and the uniform bore obviated variation in drop size.
The right gland was the index of secretory effects subsequent of denervation
and the left served as the control.
J.
C. PAUL AND
J.
439
C. DAVID
The amount of glandular secretion was observed by two methods. One,
by the simple expedient of counting the drops falling from the tubing per
minute and collecting the outflow in a graduated cylinder ( tromblod, 1955a).
Latent periods of secretion were also recorded. The other method (Richins
and Kuntz, 1953) wa by placing filter paper under the end of the exteriori ed
duct. The filter paper readily absorbed any secretion.
copious secretion
would wet the paper while lesser amounts could be estimated by observation
of the diameter of the wet spot.
The cervical sympathetic and auriculotemporal nerves were prepared for
stimulation, by mean of a Harvard inductorium at 6-8 cms. with two Ii volt
dry cells in the circuit, the tirnulation being applied for 10-30 secs.
Injection of chemical substances was made into the C moral vein.
Estimation of secretion pressure was done by introducing a blunted B-D
needle (gauge 19) into the duct and reading off pressure by means of
spinal manometeric aparatus. Arterial pressures were recorded by means of
a Baumanometer.
RESULTS
1. Secretion in the ormal, intact Parotid GLaned. The salivary flow was
sludied in three unoperated macaques, (anaesthetic. Etherf embutal) the
circulation through the parotid glands being intact. The result (Fig. 3 & 4)
NORMAL
SECRETORY VALUES
IN INTACT. RESTING GLAND.
EXPT
No
SALIVARY FLOW IN Cf:../5 MIN. PERIOO
AV.SEC
RIGHT
PAESSI~.
DROPS
DUCT
LEFT DUCT
CC'/5Mi•• DROPS
No. I
12
0.8C.C
15
No.2
I~
0.9C.C
10
N.3
12
o·ec.c
I
r
BLOOD
PIlES~
C.C'/SM-. CMS.., SAL 1104101 HG
4O·60c.,
70158
0.5C.C.
50-70CII5
88/SO
0·6 C.C.
~O'SOCIiS
I.OC.C
70/50
Fig. 3
confirmed that the two sides were secreting at similar rates, that there was no
significant difference due to either disease or obstruction.
Electrical stimulation of sympothetic nerves. Stimulation of the auriculotemporal nerve in the undisturbed gland elicited marked secretory activity,
commencing after 15-30 seconds, being greatly increased during the period of
stimulation and declining thereafter.
440
DENERVATED PAROTID GLAND
Stimulation of the peripheral end of the cervical sympathetic showed no
significant change in the secretory flow, even when prolonged stimulation was
applied. If stimulation of both sides was carried out concurrently there was a
marked decrease in salivary flow.
UUCT OF EUCnlICAl. STI......TION AND DRUGS IN ) NORMAL
UNOPERATED MACAQUES.
U".....
[LlCTAICAL
STIMULUS
PMA$YM.(I) CERVtCAl (2)
SALIVA.'"
OUT FLOW·'
]I
3D :to. 4'
2·8)
S.S:I:().13
(S-A)
a'i:O-6
.!II
1.8*0.2
J.2)
.[SoT.ICYI'D
, • 2
0.6*01 1-$*<>2
("2)
("5)
...
0..*03
(i.5)
t>9ao..
)
1·0 to...
(1.0)
O·S~
(U,
ACH
PAAA5nl nwM.THETC
(1I·t)
().,a.o.4
(
CHEMICAL
STI'" Ul.AT ION
INTACT
IN.OCIO SUP"-Y
...
,.0
-
r..5)
(S-sJ
1·2,*0"
(
• DROPS
)
().3 :t().t
(5'5)
ADA
""1Jf ICXIJ'f
~
NOR- PILe.
AD.
2><
.., ......iNt..•
(SO)
."-
N_
(SO) ()-.)
.~
. J _ ~t<>.
( •.0) (5.')
IO.o~
. .t . . I~o-'" J.UOoI
5·'
...
0-5
-
(A 5)
(A.IJ
(S-O)
ATA
.....
IOCl'7 -.,.<au
(A .•)
<>1
(A:5)
Fig. 4
When a bulldog clamp was placed on the external carotid artery, the
secretory flow was unaltered during parasympathetic stimulation but was
decreased during sympathetic stimulation. Hence reduction in blood supply
to the gland diminishes the effective secretory rate. This is in accordance
with the findings of (Coats et ai, 1956; Dirnhuber & LO\Tatt Evans, 1954;
Carlson & Mclean, 1908.
Effect of drugs. Acetylcholine in doses of 50JLg. evoked a copious secretion but the rate and amount of flow varied in the animals. There was no
increase in the secretion with larger doses. The response of both parotid
glands in the same animal remained constant. Eserine, 1 mg. a reversible
anticholinesterase, 1mg., potentiated the secretory response in both glands.
Adrenaline and or adrenaline did not produce any significant change.
In two animals ther.e was a reduction of the flow. Pilocarpine 2-4 mgs.
caused a prolonged increase in secretomotor pressure and in outflow. Atropine
0.5-1 mg. completely arrested the salivary flow due to the above drugs (Ach,
Adrenaline, Nor adrenaline & Pilocarpine).
2. Post Denervatiotl Secretory Response. The right auriculo-temporal nerve
was sectioned in six adult macaques. Preoperative stimulation of salivary
flow was done by cannulating with polythene tubing and counting the drops
for a definite time interval. Final experiments were performed after a fortnight. The average result obtained from the animals showed that decentralisation did not affect the total outflow, but the denervated side appeared to be
more sluggish than the normal side. The latent period between individual
drops was sometimes prolonged to 30-60 sees.
J.
C. PAUL AND
J.
C. DAVID
441
Response of the denervated parotid gland to electrical stimulation. It is a well
established fact that supersensitivity to adrenaline occurs after section of the
chorda in the submaxillary gland. The same occurs in the denervated parotid
gland. Stimulation of the auriculotemporal nerve produced no change in the
secretory outflow, whereas stimulation of the cervical sympathetic for 10-20
sees. showed a lag phase, then a rapid flow of drops, and finally after about
2 minutes a return to the basal state.
Emmelin (1951) has defined that supersensitivity developing in denervated salivary glands may be divided into certain categories:
1.
Threshold to stimulating substance is lowered.
2.
Longer duration of secretion.
3.
A higher secretory rate as compared with the normal.
The action of the drugs will therefore be examined by virtue of their
ability to accomplish Emmelin's postulates. The relative sensitivity of the
operated and intact glands to ACh, adrenaline and nor-adrenaline and
pilocarpine was examined.
In the case of ACh the denervated parotid gland was affected by a lower
threshold of ACh than the normal gland. The post ganglionically denervated
gland secreted faster than the normally innervated gland in the same interval
of time. (Fig. 5 & 6). The threshold of secretory stimulus varied from
l-5j.Lg., but when larger doses about 100 flog. (stimulus for the normal gland)
were given the secretory sesponse was abolished. Hence supersensitivity to
ACh was predominant in lower concentrations but large doses of ACh
appeared to produce a secretory blockade.
The denervated parotid exhibited a marked supersens!tlvlty to the
secretory effect of adrenaline, noradrenaline at low dosage ranges. Threshold
was lowered in all cases ( see fig. 7 ) and subthreshold doses gave maximal secretory responses. Secretory effects developed more slowly in the
control gland but lasted for a much longer period. This may possibly be
due to the denervated gland being more easily fatigued or damaged.
FIG. 7. SECRETORY RESPONSE OF DENERVATED RIGHT PAROTID GLAND AN
CONTROL GLAND. SALIVARY VOLUME IN
CC. TIME I MINUTES. (D") THRESHOLD DOSE OF CHEMICAL STIMULUS. (TD)
EX".
No
I
I
THOESHoLD
DOSE
DURATION
0"
I
2
DURG·
.,
NORMAL
OENERV
TO
0"
-
1.3
0.3
50J-Lg 5.0
lOOJ-Lg 5.2
lOJ-Lg 4.9
5.0
0.9
0.5
50J-Lg 5.0
IOOJ-Lg 5.2
lOJ-Lg 5.0
3
"
5.2
0.8
0.2
5
6
"
50J-Lg 5.2
lOOJ-Lg 4.9
IOJ-Lg 5.5
.
4
5.0
1.2
0.7
50J-Lg 4.9
I00J-Lg 5.0
IOJ-Lg 5.0
"
5.3
"
5.0
I. V.
Nor.Adrenaline LV.
1
IOJ-Lg 5.2
5.3
Adrenaline
I
------ -HO
Acetyl Choline. LV.
0.7
0.4
50J-Lg 5.2
IOOJ-Lg 5.0
lOJ-Lg 4.9
2.2
0.9
50J-Lg 5.3
IOOJ-Lg 4.9
NORMAL
D£NERV
--
--
3.2
6.8
2.9
1.9
6.2
5.8
1.2
1.8
0.7
5.4
6.7
6.0
0.8
8.5
3.9
3.8
4.2
6.9
0.8
0.3
3.8
3.4
1.0
2.0
3.1
2.2
8.2
1.8
7.3
6.5
2.4
9.4
-
TO
NOR·
HAL
0"
DEMERV
To
I
0"
-- - -- --,-4.8
5J-Lg 5.0 4.8 lOJ-Lg 5.0
lOOJ-Lg 5.0 15.0 0.3 5J-Lg
lOOJ-Lg
5J-Lg
IOOJ-Lg
5J-Lrt;
lOOJ-Lg
5J-Lg
I00J-Lg
5J-Lg
-IOOJ-Lg
5.0
5.2
13.8
4.9
4.8
10.7
5.0
5.0
11.2
5.2
5.2
14.5
5.0
4.9
15.8
0.2
-
0.2
-
0.3
0.3
0.4
0.2
0.7
1.2
1.0
I
I
3.2
4.4
5.2
2.9
3.2
1.7
3.3
2.8
2.5
1.7
2.9
1.3
4.8
3.9
1.2
NORMAL
DEHrRV
--
--
0.4
3.9
20
-
IOJ-Lg
5.2
0.3
0.9
2n
lOJ-Lg
4.9
-
4.2
2u
IOJ-Lg
5.0
1.4
4.8
2u
lOfLg
5.0
0.2
4.6
2rr
IOJ-Lg
5.2
2.2
2.8
2n
I
I
J.
C. PAUL AND
J.
"'--1)
~
~
10
443
C. DAVID
+--+
BASAL SECRETION· NORMAL GLAND
BASAL SECRETION
DENERVATED
10~t)NORMAL
",eN(
ACN (10,(.1.
11
DENERVATED
STiMULA TE AURIC. TENP. NERVE
9
:;ECRETlDN AfTER ELESTiw.
U
U
8
~
7
;:
0
~
u..
)0-
6
5
0:
00:(
>
4
...J
00:(
(/I
3
------------
2
_____.
...
3
2
"" . --...- ... _,-_ ..... " -j ------.; -- --
4
1
8
-'t-----i •
9
10
t
11
12
TIME IN MINUTES
SASAL SECRETORY RESPONSE IN INTACT AND DENERVATED GLAND
Fig. 6
10.
,
STUIULAn
llT.AURICULO
TEMPORAL
IN INTACT
GlAND
R
snMuun:
RT.AUR.TEMI'.
NERVE.
tl£NEIl>
VATED
RIGHT
PAROTID
l
5.6
DURATION MINutt VOLUMe
SECRETORY RESl'ONSE OF RIGHT AND LEfT PAROTID GaIl!>IN C.C.'MlN. lAVER)
Fig. 7
GLAND
444
DENERVAT2D PAROT:D GLAND
D:SCUSSION
Secretion is a primitive function of the cell. The recognition of secretion
as a functio:1 of the nerve cell in the release of neurohumoral agents such as
acetylcholine lends support to this idea. The phenomena of supersensitivity
and augmented secretion have been the basis for much speculation. In view of
the conventional concept of apparent antagonism of sympathetic and parasympathetic functions of organs, no convincing evidence has been forth coming about the activity of one type of nerve augmenting that of the other.
Earlier experiments on the secretory effect of stimulating the sympathetic
nerve to the parotid gland have given controversial results. From recent
histochemical findings it is known that some secretory cells are continually in
action while in other secretory cells individual phases of activity predominate.
Garven (1957) suggests that in salivary glands the parasympathetic fibres are
in direct relation to the mucous cells and are both hydrelatic and ecbolic to
them.
I t is generally accepted that ACh is liberated at nerve endings peripheral
to the para-sympathetic ganglion cells. Under normal conditions the specific
ChE maintains the ACh concentration at an optimal level maintaining the
gland in a state of subliminal excitation, but does not permit secretion to
ensue (Dirnhuber & Lovatt Evans 1954). P~eudo ChE has a general
"mopping up" of Ach function of a subsidiary kind. It has been seen from
the results that increasing concentrations of ACh produce secretory blockade.
This is more apparent in the denervated gland. Graham & Stavraky (1953)
have also shown that doscs of ACh above the threshold value cause vasoconstriction with diminished secretion. The denervated gland exhibits supersensitivity to ACh at low dosage levels only, and the secretory activity of the parotid gland appears to be mediated predominantly through parasympathetic
cholinergic fibres, the adrenergic fibres playing a minor role.
Vascular isolation of the parotid gland, slows down the rate of secretion
particulary during sympathetic stimulation. This may be explained by the
fact that sympathetic influences are motor to the myo-epithelial cells,
are subserved by vasomotor nerves and hence would result in vasoconstriction.
Similarly an injection of adrenaline, by increasing oxygen usage of the gland,
may cause secondary anoxia thus producing an adverse secretory effect.
However the paradoxical enhanced response to adre:.1aline and noradrenaline
given intravenously after denervation, can be explained by the generalised
rise in arterial pressure overcoming the vasoconstrictor action in the gland.
There may also be changes involving the contractile elements of the gland.
Stromblod (l955b, 1956) has proved that a supersensitivity to ACh after postganglionic denervation is because of the decrease of ChE in the gland, and in
the case of adrenaline-nor adrenaline a decrease of amine oxidase may be
responsible.
J.
C. PAUL AND
J.
C. DAVID
445
Recent anatomical evmel1ce has shown that the secretory cell has a very
definite polarity. The basal S"lu-face is the intake or receiving side, and the
luminal surface the out put or discharging side. The intermediate or basement membrane mav be depolarised on ~)lle surface by adrenaline and on
the other surface by ACh.
SUMMARY.
Parasympathetic stimulation of the parotid gland in monkeys elicited
copious secretion. The secretory response on the denervated side was sluggish,
but the total basal secretion remained unaltered. The normal spontaneous
flow of saliva is caused by a peripheral action and is maintained by a threshold
level of ACh. When the threshold ACh is exceeded, automatic secretory
blockade is demonstrable in the gland, the effect being enhanced by denervation. The denervated gland responds by exhibiting a supersensitivity
to ACh, adrenaline, nor-adrenaline and pilocarpine. This phenomenon
has been attributed to a decrease in the specific ChE content oramine oxidase
content of the denervated parotid gland.
ACKNOWLEDGEMENT
We are grateful to Dr. D. L. Graham, Professor of Anatomy, for the
valuable help she has given and to Mr. Gnanamuthu and Mr. Uthirianathan
for help with typing of the manuscript and photography.
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3.
4.
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