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Sweat Response to Acetylcholine1
SWEAT RESPONSE TO ACETYLCHOLINE' DAVID KAHN, M.A., M.D., AND STEPHEN ROTHMAN, M.D. From the Section of Dermatology, Department of Medicine, Univer8ity of Chicago, Chicago, Ill.
(Received for publication November 9, 1942) INTRODUCTION
It is generally accepted that acetyicholine acts directly on the sweat gland cells independently of the presence of a nerve supply. However, Coon and Rothman (4) in studying the local sweat response to intradermal injections of acetylcholine observed a failure of this response in five patients who had recently
undergone sympathectomy. Because this finding is difficult to explain on the basis of the present concept of the direct action of acetyicholine on sweat glands, further investigations have been carried out.
Previous experiments (29) have indicated that intradermal acetyicholine produces sweating in two ways: directly by its muscarine-like action on the sweat gland cells, and reflexly by its nicotine-like action on the nerve fibers. The latter action is effected through an axon reflex involving the terminal branches of the postganglionic sympathetic fibers supplying the sweat glands. This reflex is stimulated by small concentrations of acetylcholine (1:10,000 to 1:80,000) and paralyzed by large concentrations (1: 1000). In this paper we shall deal exclusively with the direct action of the drug on sweat gland cells. In our experiments, by the routine administration of acetylcholine in a large concentration (1: 1000), the reflex action of the drug could be
abolished without disturbing its direct action on the sweat gland cells. The reflex action is also eliminated by sympathectomy. EXPERIMENTAL
Sweat responses in humans Procedure. Acetylcholine bromide was used in a 1:1000 concentration, unless otherwise specified. Freshly prepared solutions were used, because of the insta-
bility of the acetylcholine. Normal saline was the usual diluent; the more irritating triple distilled water was used intradermally in some cases without influencing the sweat response. A volume of 0.1 to 0.2 cc. of acetylcholine bromide was injected intradermally, the usual amount being 0.12 cc. (2 minims). The sweat secretion was visualized by Minor's starch-iodine method (26). An
iodine solution2 is painted over the area to be tested. After it has dried, fine 1 Read before the Fifth Annual Meeting of the Society for Investigative Dermatology, Atlantic City, N. J., June 9, 1942. 2 The iodine solution has the following composition:
1.5 to 2 gm.
Iodine (chemically pure) Castor oil Absolute alcohol
10 cc. to 100 cc. 431
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rice starch powder is applied. Moisture causes this mixture to turn bluish black so that sweat droplets can be visualized as dark spots. The response usually described to intradermal acetyicholine consists of sweat droplets which appear on the injection wheal, often within 5—10 seconds after in-
jection. This direct muscarine-like action of acetylcholine reaches its full intensity in about five minutes, corresponding to the slow diffusion of the drug. The maximum spread may reach a diameter of 2 or 3 centimeters. Accompanyiñg the sweat reaction is a localized pilomotor response (29) which is not discussed in this paper. In the present work the local sweating response was classified as negative (0),
doubtful or very slight (±), slight (+), moderate (++), moderately strong (+++), or very strong (++++). A very slight response indicates a few tiny barely visible droplets, pinpoint in size, scattered over or adjacent to the injection wheal; a very strong reaction is characterized by confluence of large
OOJ
Negative
o
Doubtful very light
Slight
Moderately
Very etrong
+
J..: Joderate ++
Itrong +4+
+ff
Fio. 1. CLASSIFICATION OF SWEAT RESPONSES TO INTRADERMAL ACETYLCEOLINE
droplets to form a fairly diffuse or mottled blackened area over the wheal and several millimeters of surrounding skin (figs. 1, and 2). Unless otherwise designated, the tests were performed en the flexural or lateral aspects of the arm or the flexor surface of the forearm. Patients who underwent sympathectomy were tested in the supine position. Most tests in control subjects were perfdrmed with the person seated and the forearm in a horizontal position; some children were tested in the supine position. Results. The sweat response to acetylcholine injected intradermally was studied in 11 patients with partial or total sympathectomies. Tests were also carried out in 108 control subjects. Sympathectomy wasperformed by Dr. K. S. Grimson, of the Dept. of Surgery, in 10 patients with hypertension and in one patient with scleroderma. In all cases, the denervated area in which the sweat response was tested had been deprived of both the preganglionic and post-ganglionic autonomic nerve supply. The usual operative procedure involved a removal of the stellate and seven
SWEAT RESPONSE TO ACETYLCHOLINE
433
thoracic ganglia, the splanchnic nerve and part of the coeliac ganglion. In some cases the resection was carried downward to include the first two lumbar ganglia or upward to include the inferior cervical ganglia. 1. Sympathectomized male patients with hypertension. A total of 126 tests were performed on 6 sympathectomized men with hypertension (see tables 1—4). Case 1. V. M., a man aged 28, with hypertension, underwent a right thoracic paravertebral sympathectomy, followed in three weeks by a similar operation
Fia. 2.
STRONGLY POSITIVE SWEAT RESPONSE TO ACETYLCHOLINE (VISUALIZED BY MINOR'S METHOD)
Case 1. DATE
10/7 /41 11:45a.m 10/7 /41
3:15 p.m
10/8
10/8
10/10
10/28
12:40 p.m
10/28 10/30
3:30 p.m
4/20/42
RIGHT ARM
Right symp.
+ +++ ++ ++ 0
10/21
11/11
TABLE 1 Male hypertensive LEET ARM
THIGH (coNTRoL)
+++ ++++ ++++ ++++ ++++ Left symp.
0
----
0 0
on the left. The stellate and thoracic ganglia to the level of the first and second lumbar vertebrae, the splanchnic nerves and most of the coeliac ganglia were removed. The local sweat responses to intradermal acetyicholine are recorded in table 1.
Case 2. D. M., a 42 year old man with hypertension, had a left thoracic paravertebral sympathectomy followed by a similar operation on the right side 19
days later. Bilateral resection of the stellate and thoracic ganglia down to the
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level of the second lumbar vertebra was performed. In addition the right splanchnic nerve and a large portion of the coeliac ganglion was removed. The sweat responses are recorded in table 2. Case 3. P. L., a man aged 40, had a bilateral sympathectomy for hyperten-
sion. The first operation was performed on the right side; the opposite side was denervated 23 days later. The resection was carried out as in Case 1. The sweat reactions are shown in table 3. Case 4. J. M., a hypertensive, aged 31, had a left thoracic paravertebral sympathectomy, as in the first case. Table 4 records his sweat responses. Case 5. E. R., a man aged 40, had a bilateral thoracic sympathectomy for hypertension. Five months after the operation on the left 1:1000 acetyicholine TABLE 2 Case 2. Male hypertensive DATE
8/1 /41 8/5 8/7 8/9 8/11 8/14 8/16 8/18 8/19 8/19 8/19 8/21 8/25 8/27 8/29
9/8 9/16
10:30 a.m 2:30 p.m 5:15 p.m
RIGHT ARM
++++ ++++ ++++ ++++ ++++ ++++ ++++ Right symp. ++++ ++ ++++ ++++ +++ ++ ++
THIGH (CoNTRoL)
Left symp.
++± ++++ +± +++ 0 ++±
++±+ + ++++ +
0
----0
0
0
10/3
4/1 /42
LEFT ARM
+++ ++++ +++ ++++ ++++
injected intradermally gave a negative sweat response on the extensor surface of the left arm. A 1:200 concentration of the drug caused a very slight response.
Case 6. T. M., a man with hypertension, underwent an abdominal sympathectomy, a right thoracic and a left thoracic sympathectomy at intervals of
approximately 3 weeks. On the 8th and 10th day after the resection on the left, very slight sweat responses were obtained on the 1:200 acetyicholine. The right thigh, which served as a control, gave a moderately strong acetyicholine response; heat sweating was also present in this area.
In all male patients who were tested prior to operation, the initial sweat response to acetylcholine was strong or very strong. In those cases in which the first tests were performed after unilateral sympathectomy had been done, the response of the unoperated side, which served as a control, was likewise strong. After operation, there was a variable diminution and delay in the response
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SWEAT RESPONSE TO ACETYLCHOLINE
to acetyicholine of the denervated side. The earliest observed diminished response was 3 hours after operation (table 5). Subsequently, in all six cases, consistently negative or very slight responses were observed beinning from 2 to 62 days after sympathectomy. In the 3 cases observed six to eight months after operation the sweating response was still absent or weak. In four cases, eight TABLE 3 Case 3. Male hypertensive DATE
8/13/41
8/15 8/16 8/16 8/17 8/18 8/19
11:OOa.m
1:00 p.m
8/21 8/27
9/8 9/8 9/9 9/16 10/3
LEFT ARM
++++ ++++ Right symp. ++++
++++ ++++ ++++ ++++ ++++ ++++ +++ ++++ ++++ ++++ ++++ Leftsymp. ++++ ++ ++++
++ ++ ++ +++
++ +
8/23 9/4 9/7
RIGHT AJi
0 0
11:30a.m 3:00 p.m 4:30 p.m E 0 0 0
4/ 7/42 4/19
THIGH (co&ToL)
++++ ++++ ++++ ++++
0
0 0
TABLE 4 Case 4. Male hypertensive DATE
4/1/42 12:15 4/7 2:30 4/7 5:15 4/7 4/8 4/9 4/11 4/19 5/19
RIGHT ARM
+++ ++++ ++++ ++++ ++++ ++++ ++++ ++++
LEFT ARM
++++ ++++ ++++ +++
Left symp.
++ ++ 0 0
parallel tests with 1:1000 mecholyl (acetyl-beta-methyicholine hydrochloride) were also performed; the results were similar. In each case, when a comparable control was used, the intact side gave a strongly positive sweat reaction. After bilateral denervation o the sympathetic supply to the upper extremities, the anterior surface of the thigh proved to be a fairly satisfactory control site. In a more complete sympathectomy, where the
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thigh was partially denervated, its sweat response could be used for a rough comparison with that of the totally sympathectomized upper extremities. 2. Male patient with scleroderma. In one man with generalized scieroderma 88 acetyicholine tests were performed over an 11 month period after sympathectomy. There was a diminution inthe sweat response as in the previous cases. Case 7. In J. F., a 48 year old man with scieroderma, the stellate and upper 7 thoracic ganglia and the upper portion of the greater splanchnic nerves were excised bilaterally. The right inferior cervical ganglion was also removed The operation was performed in two stages 14 days apart. The sweat responses are recorded in table 6. In this case the sweat response to acetyicholine was tested for the first time
19 days after right sympathectomy and 4 days after denervation on the left. There were no control observations before the operation. Almost daily tests were
performed during the first month of observation, throughout which time the TABLE 5 Summary of results insympathectomized male hypertensives CASE NO. 1
2
34
64
3
4
264
48
5
6
Earliest observed diminished response after sympathectomy in hours Consistently negative or very slight responses (days after sympathectomy) RIght
14
27
2 Left 62 Duration of observation period after 64 mo. 8 mo. sympathectomy
18
25
12
8 mo.
14 mo.
8
54 mo.
10 da.
sweat responses were usually diminished or absent, though not consistently so.
During the 2nd month of observation the response to acetyicholine became consistently strong on the left arm and the patient also noted the return of sweating caused by heat in this area. The latter observation was confirmed by a heat sweating test which indicated the return of nerve function in this area with consequent ability to perspire upon exposure to ordinary heat. Subsequent observations showed diminished responses. Thus, oscillations in the response of the sweat glands were noted over an 11 month period following sympathectomy. In some of the experiments, when a weak or negative response to acetyicholine
resulted, the test site was cleansed with alcohol and the iodine-starch mixture reapplied over the injection wheal. The second application, which was usually made 5 to 10 minutes after the injection, frequently was followed by a slightly stronger sweating response than was noted with the original test. No explanation is offered for this finding.
437
SWEAT RESPONSE TO ACETYLCHOLINE
3. Female patients with hypertension. Fifty-one tests were performed on four
patients. In these women, the diminished sweat responses to acetyicholine after sympathectomy were similar to those in men. However, the control areas TABLE 6 Man with scleroderma LERT ARM
DATE
RIGHT ARM
6/23/41
Right symp. Left symp.
7/7 7/11
7/12 7/14 7/15 7/16 7/17 7/18 7/19 7/21 7/23 7/24 7/25 7/26 7/28 7/29 7/30 7/31
0
+ +
0
+++? +
++ 0 0
0
0 0 0 0
0 0
++++ 0 ++ ++++
+
++++ 0 0
0 0
++
8/7 8/11 8/21
0 0 0
9/4
+
+
8/4 8/5
10/2 5/19/42
THIGH (coNTRoL)
++ ++++ +++ +++
+ +
+
++ ++++ ++ ++ ++ + ++++ +++ +++ ++++
TABLE 7 Local sweat response to intradermal acetyicholine (1:1000) in 100 control subjects
Men Women
NEGATIVE TO SLIGHT RESPONSE
NUMBER OP SUBJECTS
MODERATE TO VERY STRONG RESPONSE
59 41
56 (94.9%)
3 ( 5.1%)
4 ( 9.8%)
37 (90.2%)
usually gave relatively weak responses also. Therefore, no detailed data are presented on these cases.
4. Control subjects. Because of the unexpected difference in the sweat response noted in men as compared with women in normal areas of skin before sympathectomy, 152 tests were performed in 100 adult control subjects. Table 7 records the sweat responses of 59 men and 41 women.
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In 55 men tested with acetyicholine and 4 tested with mecholyl, the sweat response was moderate, strong or very strong in 56 instances (94.9%); in only 3 cases (5.1%) was there absent or very slight sweating. In 40 women injected with acetyicholine and one tested with mecholyl, the • sweat response was moderate to very strong in only 4 cases (9.8%); a negative or slight response was present in 37 subjects (90.2%). In some cases the tests were repeated after a varying number of weeks. Of ten controls, three men, whose initial response was weak, subsequently showed moderate or strong reactions. Because of this sex difference in response, the possibility of hormonal influences presented itself. Hence, a small group of children below the age of puberty were tested. In 4 boys ranging in age from 3 to 11 years, the sweat response was strong in 2 cases and negative in 2 cases. Four girls, 5 to 9 years of age, showed negative or very slight responses in 2 cases, and moderate or strong reactions in two. Many of the subjects for whom only acetyicholine responses are recorded in Table 7 were also tested with mecholyl. 43 tests were made in 39 subjects.
Parallel injections of the two drugs gave similar results. In some cases the mecholyl response was slightly greater than that with acetyicholine, but this was
not a constant finding. In 3 cases 1:1000 pilocarpine hydrochloride was injected with comparable results. Sweat responses in cats
Procedure. The sweat response to acetyicholine was also studied in the eat's paw in 3 animals. The appearance of sweat droplets was observed through a magnifying glass. Denervation of the sweat glands of the footpads was achieved
by section of the sciatic nerve. For each sweat test, 0.1 to 0.2 cc. of acetylcholine was injected subcutaneously into footpads of cats under ether anesthesia.
In each of the three cats the sweat response on the denervated side was first observed to become persistently negative or weak in 1, 5, and 13 days respectively, as compared with strong responses on the intact side. In one animal, the denervated paw gave a slightly stronger response than the normal paw for the first 24 hours, after which the reaction of the operated side became progressively weaker. The use of 1:200, 1:1000 or 1:5000 dilutions gave es-
sentially similar results. After sciatic nerve section, pallor was noted in the toe pads of the operated side. When a weakened and delayed sweat response after acetyicholine was obtained, the delay appeared to correspond to the time required for the pale toe pad to become reddened after injection. To test the assumption that the diminished sweat response to acetyicholine
after denervation might reflect a decrease in permeability of the sweat gland cell, the technique of vital staining was employed. Neutral red was found to be a suitable selective stain for sweat g'ands. The following mithod was used: The cat was anaesthetized with ether. 0.5 cc. of 0.5% aqueous solution of neutral red was injected into the footpad of the cat's paw. The footpad was excised 8 minutes after injection and placed in
SWEAT RESPONSE TO ACETYLCIIOLINE
439
formaldehyde. Frozen sections, 10 micra in thickness, were prepared, alcohol being used in the process. The tissue was mounted in glycerine. Histologic examination of the footpads of four cats failed to reveal any consistent difference in the degree of staining of denervated sweat glands as compared with intact glands. In general, the results were too variable, the technique too uncertain, and the number of animals too small to draw any definite conclusions at present regarding a change in permeability of the sweat gland cells after denervation. Further experimental work is in progress. DISCUSSION
1. Diminution in the sweat response to acetyicholine after sympathectomy
Investigators have indicated that the innervation of the sweat glands, although anatomically sympathetic, is pharmacologically parasympathetic. This is explained by the concept of two sets of postganglionic fibers in the thoracolumbar (sympathetic) system, adrenergic and cholinergic. 1) On stimulation of adrenergic fibers, at their endings sympathin, an epinephrine-like principle is liberated. The effect of adrenergic nerve impulse is identical with the effect of injection of epinephrine. Both effects are negated by ergotamine. 2) The cholinergic fibers liberate acetylcholine at their endings on stimulation. The effector cells are stimulated also by acetylcholine, pilocarpine, physostigmine,
and other parasympathetic drugs, and paralyzed by atropine. The fibers supplying the sweat glands are cholinergic although anatomically they course with the sympathetic chain, which otherwise contains adrenergic fibers only. In its direct action on sweat glands, acetylcholine conforms with the concept of a chemical mediator. According to the neurohumoral theory, the chemical mediator is liberated at the nerve ending and acts directly upon the effector organ. According to this theory, if the chemical mediator acetyicholine were injected directly into the gland-bearing area, a local sweating response would be expected also after denervation. Instead, our experimental results indicate that the sweating response becomes weak or absent after nerve section. Similar paradoxical behavior has been described for the parasympathomimetic drug, pilocarpine. There has been much controversy regarding the action of pilocarpine on sweat glands because many investigators have reported the disappearance of the sweat response to pilocarpine after denervation. Physiologists on the basis of Langley's (16) and Burn's (2) experiments hold that pilocarpine acts directly on the sweat glands, independent of the presence of a nerve supply. Clinical neurologists (20) question this concept and have advanced various theories of central and peripheral nervous action of the drug. Recent evidence (12) which attempts to explain this confusion indicates that the pilocarpine sweat response will disappear only after complete degeneration of postganglionic neurons supplying the sweat gland. However, the possibility of a direct action on the sweat gland cell by high concentrations of pilocarpine is admitted. In any case, it has frequently been observed that if sufficient time has elapsed after denervation, the sweat response to injected pilocarpine disappears.
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THE JOURNAL OF INVESflGATIVE DERMATOLOGY
The weak or negative responses to acetyicholine of the denervated sweat glands represent an exception to the general rule of sensitization, as set forth by Cannon (3). As a general rule, smooth muscle and glands that are innervated by adrenergic and cholinergic nerve fibers give a greater response when chemically stimulated after denervation, especially if the chemical mediator, epinephrine or acetyleholine, supplies the stimulus. This has been explained by Cannon as a sensitization phenomenon, in which an increased irritability to chemical agents develops in the structure. Applying this principle to smooth muscle, Shen and Cannon (30) found increased sensitivity of the denervated iris to acetylcholine as compared with the normal iris. Simeone and Maes (32) noted similar results in submaxillary glands, to which the sympathetic nerves had been severed. However, when the parasympathetic innervation to the submaxillary gland was removed by severing the chorda tympani (mainly a preganglionic denervation), the expected increased salivary response to acetylcholine did not develop when tested two months after denervation, as noted by Pierce and Gregersen (29). Instead the drug apparently exerted a depressant influence, a result at variance with the concept of sensitization. Pilocarpine caused an increased response in this experiment. A second exception to Cannon's rule is represented by our experiments, in which the denervated sweat gland showed a decreased or absent response to acetylcholine instead of the increase to be expected were the structure "sensiti-
zed." It might be anticipated that acetyicholine would cause an increased response in the sweat gland after denervation. Instead, the response was diminished or absent after sympathectomy in our patients. In the experiments on cats, one animal showed a transitory increased response after denervation, followed soon by a decrease. In man, diminished sweat responses to intradermal acetylcholine were observed as early as 3 hours after sympathectomy in case 1. and 6 hours postoperatively in case 2. After a period of oscillation varying from 2 to 62 days, these responses became consistently negative or very slight and remained so during the entire period of observation—in some cases 6 to 8 months.
In all tests a concentration of the drug strong enough to paralyze its reflex action was used in order that the direct action of acetylcholine could be studied. Thus the diminished sweat response after sympathectomy is not to be explained on the basis of denervation of the reflex arc.
That sweating produced by intradermal acetyicholine is not due to simple mechanical squeezing out of sweat droplets from the gland by the pressure due to the volume of the injected drug is shown by experiments with saline controls. Langley (16) and Langely and Uyeno (17) injected Ringer's solution and ob-
tained a slight transient sweat secretion in the cat's footpad. This response
could be easily differentiated from the more profuse and more lasting response to pilocarpine. Similarly Coon and Rothman (4) noted slight or absent sweating with saline controls as compared to strong responses to acetyicholine. When
present, the reaction to saline consisted of sweat droplets limited to a small
SWEAT RESPONSE TO ACETYLCHOLINE
441
part of the footpad, whereas the response to the same volume of acetylcholine solution generally involved the whole surface of the injected toe pad. It might be argued that the concentration of the drug (1:1000) was not at the optimum level for direct stimulation of the sweat gland cells. This possibility is suggested by the observation of Myerson and co-workers (27) who noted that a greater concentration of mecholyl, when injected intradermally, is needed in fall than in summer to produce a good sweat response. This objection is answered by the fact that the control area gave strong responses to this concentration. In addition, some tests were repeated using a stronger concentration (1:200) of the drug with the same results. Acetyicholine is an unstable preparation, being destroyed rather rapidly by
the cholinesterase present in the tissues. To check the possibility that the weak or negative sweat responses might be due to this factor, parallel tests were carried out with mecholyl (acetyl-beta-methyicholine), a relatively stable derivative of acetylcholine, since it is only slightly hydrolyzed by cholinesterase. The
results with mecholyl were comparable to those with acetylcholine. In some cases, the response to mecholyl was slightly greater than that to acetylcholine, but not constantly. In view of the weak or negative reactions to acetylcholine after sympathectomy, it might be assumed that degneration of the sweat gland occurs relatively soon after denervation. However, the rapidity of disappearance of the sweat response would, in itself, militate against such a supposition. Furthermore, when we consider that after the development of a negative sweat response, oscil-
lating reactions are subsequently observed, it is evident that the denervated sweat glands are still functioning. That this early degeneration does not occur is attested by the following evidence: 1) Histologic study of skin obtained both before and after denervation of an area fails to show degeneration of the sweat glands following sympathectomy, according to a recent report (7). 2) Excessive local heating of a denervated area of skin after sympathectomy will evoke sweat-
ing in this region, even though ordinary heat sweating is not observed. The direct local response of the sweat glands to excessive heat was observed by Kuno (14) in cats 3—6 weeks after sciatic nerve section. 3) A type of response desig-
nated as "contact sweating" has been observed up to a year and a half after postganglionic sympathectomy. For some unexplained reason, when two sympathectomized areas of skin are placed in contact in a warm atmosphere, the corresponding sweat glands will secrete, according to Hyndman and Wolkiri (11). However, these authors do not distinguish sweating from insensible perspiration, which occur in such areas independent of sweat gland function (10). Thus the problem remains unsolved. We may explain the failure of a denervated area to sweat upon exposure to heat by the fact that heat sweating results
from a nervous impulse. Thus, after sympathectomy, there is no impulse to liberate acetyicholine at the nerve ending and as a result there is no activation of the sweat gland. However, when the "chemical mediator" is supplied by intradermally injected acetyicholine which suffuses the sweat gland-containing area with the drug, it is difficult to explain why a weak or negative reaction
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THE JOURNAL OF INVESTIGATIVE DERMATOLOGV
obtains. We cannot conclude that acetylcholine does not have a direct action on sweat glands because the sweat responses are sometimes positive, even though
slight, for long periods after sympathectomy, during which time variable responses are noted. Furthermore, by producing acetylcholine sweating in a locally anaesthetized area of skin in a normal control subject, we have additional evidence that the drug acts directly on the sweat gland. A possible explanation of the diminished or absent response is that alterations in the sweat gland cell-membrane, perhaps in the nature of a decreased perme-
ability, may occur after sympathectomy, thereby inhibiting the penetration of injected acetylcholine into the cell. This possibility was tested by the technique of vital staining with neutral red, which proved to be a good selective stain for sweat glands. However, the results were inconsistent.
Thus it appears that the response to acetylcholine of the denervated sweat gland is an exception to the basic law of sensitization of the structure after denervation. 2. Sex difference in the sweat response to acetyicholine of control subjects
The tendency to a stronger sweating response of men as compared with women after intradermal injection of acetyicholine has not been recorded previouslyas far as can be ascertained. No explanation is offered for this phenomenon, although many factors may be considered. Hormonal influences seem likely to play a role in causing this sex difference in response. It may or may not be significant that two of the four women who gave a stronger response than the others were pregnant. This sex difference in response was not due to atmospheric variations, since both sexes were tested under identical conditions of temperature and humidity.
The number of sweat glands per unit area of skin may play a role in the variations noted. It has been observed that relatively more sweat glands are present on the extremities of people whose costume leads to exposure of these areas (14). Infants have relatively more sweat glands per unit area of skin than adults. The number of children tested with acetylcholine was too small for the results to be conclusive. The relative amount of adipose tissue present in an area may influence the density of sweat gland distribution. But the acetylcholine sweat response did not appear to be related to the local or general fat ditribution. Evidence indicates that the amount of sweat does not depend entirely on the anatomic density of sweat glands in an area, since maniy of the
glands may not be in a functional state, as pointed out by Jurgensen (13). According to Adachi (1) there is no marked difference between children and adults with regard to the amOunt of sweating per unit of body surface. Although all sweat glands are present at birth, all do not function.
In any case it is not a likely assumption that the sweat glands in women could be so sparsely distributed in the area tested as to fail to give a visible response when directly stimulated by the drug in sufficiently high concentration. Furthermore, definite heat sweating was present in the same areas that responded poorly or negatively to acetycholine, as evidenced frequently by history, direct observation, or the starch-iodine visualization method.
SWEAT RESPONSE TO ACETYLCHOLINE
443
There was no apparent correlation of heat sweating or emotional sweating with the sweat response to acetylcholine in the control subjects. For example, women usually had a poor response to the acetyleholine test, whether they perspired slightly or profusely in hot weather or in response to emotions. A preference for hot or cold weather bore no relationship to the sweat responsetested. CONCLUSIONS:
1. The local sweat response to intradermally injected acetylcholine diminishes or disappears after sympathectomy. 2. This finding is an exception to the rule of sensitization of structures after denervation (Cannon).
3. The local sweat response to the intradermal injection of acetyicholine is greater in men than in women.
We are greatly indebted to Dr. K. S. Grimson for his permission to make skin tests on his sympathectomized patients. Dr. E. M. K. Geiling placed the equipment of the Department of Pharmacology at our disposal and gave us valuable advice. Dr. J. M. Coon helped us greatly in the animal experiments. BIBLIOGRAPHY 1. ADACHI, J., Cited by KUNO, Y.: Functional variations in the human sweat glands. Jap. J. M. Sc., III, Biophysics, 5: 4*_5* (March) 1938. 2. BURN, J. H.: The relation of nerve supply and blood flow to sweating produced by pilocarpine. J. Physiol., 56: 232 (May) 1922. 3. CANNON, W. B.: A law of denervation. Am. J. Med. Sci., 198:737 (Dec.) 1939. 4. Cooar, J. M., AND ROTHMAN, S.: Sweat response to drugs with nicotine-like action. J.
of Pharm. and Exper. Therap.,73: 1 (Sept.) 1941. 5. DALE, H. H., AND FELDBERG, W.: The chemical transmission of secretory impulses to
the sweat glands of the cat. J. Physiol., 82: 121, 1934. 6. ELLIOTT, T. R. : Action of adrenalin. J. Physiol., 32: 401 (July) 1905. 7. GURNEY, R., AND BUNNELL, I. L.: A study of the reflex mechanism of sweating in the
human being; effect of anesthesia and sympathectomy. J. Clin. Invest., 21: 269 (May) 1942. 8. GUTTr,SAN, L., AND LIST, C. F.: Zur Topik und Pathophysiologie der Schweiss-Sekretion. Ztschr. f. d. ges. Neurol. u. Psychiat., 116: 504, 1928.
9. HAMPEL, C. W.: Effect of denervation on sensitivity to adrenine of smooth muscle in nictitating membrane of cat. Am. J. Physiol., 111: 611 (April) 1935. 10. HARE, K., AND HINSEY, J. C. : The autonomic nervous system. Ann. Rev. Physiol., 4: 434, 1942. 11. HYNDMAN, 0. R., AND WOLKIN, J.: Sweat mechanism in man: study of distribution of
sweat fibers from sympathetic ganglia, spinal roots, spinal cord and common carotid artery. Arch. Neurol. & Psychiat., 45: 446 (March) 1941. 12. HYNDMAN, 0. R., AND WOLKIN, J.: The pilocarpine sweating test. I. A valid indicator
in differentiation of preganglionic and postganglionic sympathectomy. Arch. Neurol. & Psychiat., 45: 992 (June) 1941. 13. JtJRqENSEN, E.: Mikrobeobachtungen der Schweiss-Sekretion der Haut des Mensehen unter Kontrastfarbung. Dtsch. Arch. kim. Med., 144: 193 (June) 1924. 14. KUNO, Y.: The Physiology of Human Perspiration. London, J. & A. Churchill, Ltd., 1934.
15. LANGLEY, J. N.: Observations on the physiological action of extracts of the supra-renal
bodies. J. Physiol., 27: 237 (Oct.) 1901.
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16. LANGLEY, J. N.: The secretion of sweat. I. Supposed inhibitory nerve fibers in the posterior nerve roots; secretion after denervation. J. Physiol., 56: 110 (Feb.) 1922. 17. LANGLEY, J. N., AND [JYENO, K.: Secretion of sweat; effect of vaso-constriction and of
adrenaline. J. Physiol., 66: 206 (May) 1922. 18. LIST, C. F., AND PERT, M. M.: Sweat secretion in man. I. Sweating Responses in normal persons. Arch. Neurol. & Psychiat., 39: 1228 (June) 1938. 19. LIsT, C. F., AND PEEr, M. M.: Sweat secretion in man. II. Anatomic distribution of
sweating disturbances associated with lesions of the sympathetic nervous system. Arch. Neurol. & Psychiat., 40: 27 (July) 1938. 20. Lrsr, C. F., AND PEET, M. M.: Sweat secretion in man. III. Clinical observations on sweating produced by pilocarpine and mecholyl. Arch. Neurol. & Psyohiat., 40: 269 (Aug.) 1938.
21. Ltrco, J. V.: Sensitization of inhibited structures by denervation. Am. J. Physiol., 120: 179
(Sept.) 1937.
22. MARs, J. P.: Effect of removal of superior cervical ganglion on lachrymal secretion. Am. J. Physiol., 123: 359 (Aug.) 1938. 23.
MARRAZZI, R., AND MAREAZZI, A. S.: Ionic permeability of the sensitized nictitating
membrane of the cat. Am. J. Physiol., 133: 377, 1941. 24. MELTzER, S. J., AND AURE, C. M. : Studies on the "paradoxical" pupil-dilatationcaused
by adrenalin. Am. J. Physiol., 11: 28,37 (April) 1904. 25. MENG, C. W. : Role of cholinesterase in sensitization of muscle to acetyloholine. Chinese J. Physiol., 15: 143 (April 30) 1940. 26. MINOR, V.: Eines neues Verfahren zu der klinischen Untersuchung der Schweissabsonderung. Deutsche Ztsohr. f. Nervenh., 101: 302—308, 1927. 27. MYERSON, A., LOMAN, J., AND RIN1EL, M.: Human autonomic pharmacology; general
and local sweating produced by acetyl-beta-methyl-choline chloride (mecholyl). Am. J. M. Sc., 194: 75 (July) 1937. 28. PIERcE, F. R., AND GREGERSEN, M. I.: Changes in submaxillary secretory response to
pilocarpine after
section of chorda tympani. Am. J. Physiol., 120: 246 (Oct.) 1937.
29. ROTRMAN, S., AND CooN, 30.
31.
J. M.:
Axon reflex responses to acetyleholine
Invest. Dermat., 3: 79 (April) 1940. SEEN, S. C., AND CANNON, W. B.: Sensitization
of denervated
in the skin. J.
pupillary sphincter to
acetylcholine. Chinese J. Physiol., 10: 359 (May 15) 1936. SIMEONE, F. A.: Effect of regeneration of nerve supply on sensitivity of denervated nictitating membrane to adrenine. Am. J. Physiol.,120: 466, 1937.
32. SIMEONE, F. A., AND MARS, J. P.: Sensitization of submaxillary gland by sympathetic denervation. Am. J. Physiol., 125: 674 (April) 1939. 33. SOLLMANN, T.: A Manual of Pharmacology. Philadelphia, W. B. Saunders Company, 1942.
34. YOUMANS, W. B.: Similarity of effects of adrenalin and inhibitory sympathin on intestinal motility; sensitization by denervation. Am. J. Physiol., 123:424 (Aug.) 1938.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
94
linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
24. Wynn, C. H. and Iqbal, M.: Isolation of rat
skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.
human epidermis, p. 511, The Epidermis
Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-
No warranty is given about the accuracy of the copy.
Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.
Cell Biol., 27: 603, 1965.
27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-
C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.
30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.
31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-
lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.
32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.
33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.
34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:
Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.
43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.
44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.
45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.
46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,
Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by
Res., 33: 176, 1964.
(Received for publication November 9, 1942) INTRODUCTION
It is generally accepted that acetyicholine acts directly on the sweat gland cells independently of the presence of a nerve supply. However, Coon and Rothman (4) in studying the local sweat response to intradermal injections of acetylcholine observed a failure of this response in five patients who had recently
undergone sympathectomy. Because this finding is difficult to explain on the basis of the present concept of the direct action of acetyicholine on sweat glands, further investigations have been carried out.
Previous experiments (29) have indicated that intradermal acetyicholine produces sweating in two ways: directly by its muscarine-like action on the sweat gland cells, and reflexly by its nicotine-like action on the nerve fibers. The latter action is effected through an axon reflex involving the terminal branches of the postganglionic sympathetic fibers supplying the sweat glands. This reflex is stimulated by small concentrations of acetylcholine (1:10,000 to 1:80,000) and paralyzed by large concentrations (1: 1000). In this paper we shall deal exclusively with the direct action of the drug on sweat gland cells. In our experiments, by the routine administration of acetylcholine in a large concentration (1: 1000), the reflex action of the drug could be
abolished without disturbing its direct action on the sweat gland cells. The reflex action is also eliminated by sympathectomy. EXPERIMENTAL
Sweat responses in humans Procedure. Acetylcholine bromide was used in a 1:1000 concentration, unless otherwise specified. Freshly prepared solutions were used, because of the insta-
bility of the acetylcholine. Normal saline was the usual diluent; the more irritating triple distilled water was used intradermally in some cases without influencing the sweat response. A volume of 0.1 to 0.2 cc. of acetylcholine bromide was injected intradermally, the usual amount being 0.12 cc. (2 minims). The sweat secretion was visualized by Minor's starch-iodine method (26). An
iodine solution2 is painted over the area to be tested. After it has dried, fine 1 Read before the Fifth Annual Meeting of the Society for Investigative Dermatology, Atlantic City, N. J., June 9, 1942. 2 The iodine solution has the following composition:
1.5 to 2 gm.
Iodine (chemically pure) Castor oil Absolute alcohol
10 cc. to 100 cc. 431
432
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
rice starch powder is applied. Moisture causes this mixture to turn bluish black so that sweat droplets can be visualized as dark spots. The response usually described to intradermal acetyicholine consists of sweat droplets which appear on the injection wheal, often within 5—10 seconds after in-
jection. This direct muscarine-like action of acetylcholine reaches its full intensity in about five minutes, corresponding to the slow diffusion of the drug. The maximum spread may reach a diameter of 2 or 3 centimeters. Accompanyiñg the sweat reaction is a localized pilomotor response (29) which is not discussed in this paper. In the present work the local sweating response was classified as negative (0),
doubtful or very slight (±), slight (+), moderate (++), moderately strong (+++), or very strong (++++). A very slight response indicates a few tiny barely visible droplets, pinpoint in size, scattered over or adjacent to the injection wheal; a very strong reaction is characterized by confluence of large
OOJ
Negative
o
Doubtful very light
Slight
Moderately
Very etrong
+
J..: Joderate ++
Itrong +4+
+ff
Fio. 1. CLASSIFICATION OF SWEAT RESPONSES TO INTRADERMAL ACETYLCEOLINE
droplets to form a fairly diffuse or mottled blackened area over the wheal and several millimeters of surrounding skin (figs. 1, and 2). Unless otherwise designated, the tests were performed en the flexural or lateral aspects of the arm or the flexor surface of the forearm. Patients who underwent sympathectomy were tested in the supine position. Most tests in control subjects were perfdrmed with the person seated and the forearm in a horizontal position; some children were tested in the supine position. Results. The sweat response to acetylcholine injected intradermally was studied in 11 patients with partial or total sympathectomies. Tests were also carried out in 108 control subjects. Sympathectomy wasperformed by Dr. K. S. Grimson, of the Dept. of Surgery, in 10 patients with hypertension and in one patient with scleroderma. In all cases, the denervated area in which the sweat response was tested had been deprived of both the preganglionic and post-ganglionic autonomic nerve supply. The usual operative procedure involved a removal of the stellate and seven
SWEAT RESPONSE TO ACETYLCHOLINE
433
thoracic ganglia, the splanchnic nerve and part of the coeliac ganglion. In some cases the resection was carried downward to include the first two lumbar ganglia or upward to include the inferior cervical ganglia. 1. Sympathectomized male patients with hypertension. A total of 126 tests were performed on 6 sympathectomized men with hypertension (see tables 1—4). Case 1. V. M., a man aged 28, with hypertension, underwent a right thoracic paravertebral sympathectomy, followed in three weeks by a similar operation
Fia. 2.
STRONGLY POSITIVE SWEAT RESPONSE TO ACETYLCHOLINE (VISUALIZED BY MINOR'S METHOD)
Case 1. DATE
10/7 /41 11:45a.m 10/7 /41
3:15 p.m
10/8
10/8
10/10
10/28
12:40 p.m
10/28 10/30
3:30 p.m
4/20/42
RIGHT ARM
Right symp.
+ +++ ++ ++ 0
10/21
11/11
TABLE 1 Male hypertensive LEET ARM
THIGH (coNTRoL)
+++ ++++ ++++ ++++ ++++ Left symp.
0
----
0 0
on the left. The stellate and thoracic ganglia to the level of the first and second lumbar vertebrae, the splanchnic nerves and most of the coeliac ganglia were removed. The local sweat responses to intradermal acetyicholine are recorded in table 1.
Case 2. D. M., a 42 year old man with hypertension, had a left thoracic paravertebral sympathectomy followed by a similar operation on the right side 19
days later. Bilateral resection of the stellate and thoracic ganglia down to the
434
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
level of the second lumbar vertebra was performed. In addition the right splanchnic nerve and a large portion of the coeliac ganglion was removed. The sweat responses are recorded in table 2. Case 3. P. L., a man aged 40, had a bilateral sympathectomy for hyperten-
sion. The first operation was performed on the right side; the opposite side was denervated 23 days later. The resection was carried out as in Case 1. The sweat reactions are shown in table 3. Case 4. J. M., a hypertensive, aged 31, had a left thoracic paravertebral sympathectomy, as in the first case. Table 4 records his sweat responses. Case 5. E. R., a man aged 40, had a bilateral thoracic sympathectomy for hypertension. Five months after the operation on the left 1:1000 acetyicholine TABLE 2 Case 2. Male hypertensive DATE
8/1 /41 8/5 8/7 8/9 8/11 8/14 8/16 8/18 8/19 8/19 8/19 8/21 8/25 8/27 8/29
9/8 9/16
10:30 a.m 2:30 p.m 5:15 p.m
RIGHT ARM
++++ ++++ ++++ ++++ ++++ ++++ ++++ Right symp. ++++ ++ ++++ ++++ +++ ++ ++
THIGH (CoNTRoL)
Left symp.
++± ++++ +± +++ 0 ++±
++±+ + ++++ +
0
----0
0
0
10/3
4/1 /42
LEFT ARM
+++ ++++ +++ ++++ ++++
injected intradermally gave a negative sweat response on the extensor surface of the left arm. A 1:200 concentration of the drug caused a very slight response.
Case 6. T. M., a man with hypertension, underwent an abdominal sympathectomy, a right thoracic and a left thoracic sympathectomy at intervals of
approximately 3 weeks. On the 8th and 10th day after the resection on the left, very slight sweat responses were obtained on the 1:200 acetyicholine. The right thigh, which served as a control, gave a moderately strong acetyicholine response; heat sweating was also present in this area.
In all male patients who were tested prior to operation, the initial sweat response to acetylcholine was strong or very strong. In those cases in which the first tests were performed after unilateral sympathectomy had been done, the response of the unoperated side, which served as a control, was likewise strong. After operation, there was a variable diminution and delay in the response
435
SWEAT RESPONSE TO ACETYLCHOLINE
to acetyicholine of the denervated side. The earliest observed diminished response was 3 hours after operation (table 5). Subsequently, in all six cases, consistently negative or very slight responses were observed beinning from 2 to 62 days after sympathectomy. In the 3 cases observed six to eight months after operation the sweating response was still absent or weak. In four cases, eight TABLE 3 Case 3. Male hypertensive DATE
8/13/41
8/15 8/16 8/16 8/17 8/18 8/19
11:OOa.m
1:00 p.m
8/21 8/27
9/8 9/8 9/9 9/16 10/3
LEFT ARM
++++ ++++ Right symp. ++++
++++ ++++ ++++ ++++ ++++ ++++ +++ ++++ ++++ ++++ ++++ Leftsymp. ++++ ++ ++++
++ ++ ++ +++
++ +
8/23 9/4 9/7
RIGHT AJi
0 0
11:30a.m 3:00 p.m 4:30 p.m E 0 0 0
4/ 7/42 4/19
THIGH (co&ToL)
++++ ++++ ++++ ++++
0
0 0
TABLE 4 Case 4. Male hypertensive DATE
4/1/42 12:15 4/7 2:30 4/7 5:15 4/7 4/8 4/9 4/11 4/19 5/19
RIGHT ARM
+++ ++++ ++++ ++++ ++++ ++++ ++++ ++++
LEFT ARM
++++ ++++ ++++ +++
Left symp.
++ ++ 0 0
parallel tests with 1:1000 mecholyl (acetyl-beta-methyicholine hydrochloride) were also performed; the results were similar. In each case, when a comparable control was used, the intact side gave a strongly positive sweat reaction. After bilateral denervation o the sympathetic supply to the upper extremities, the anterior surface of the thigh proved to be a fairly satisfactory control site. In a more complete sympathectomy, where the
436
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
thigh was partially denervated, its sweat response could be used for a rough comparison with that of the totally sympathectomized upper extremities. 2. Male patient with scleroderma. In one man with generalized scieroderma 88 acetyicholine tests were performed over an 11 month period after sympathectomy. There was a diminution inthe sweat response as in the previous cases. Case 7. In J. F., a 48 year old man with scieroderma, the stellate and upper 7 thoracic ganglia and the upper portion of the greater splanchnic nerves were excised bilaterally. The right inferior cervical ganglion was also removed The operation was performed in two stages 14 days apart. The sweat responses are recorded in table 6. In this case the sweat response to acetyicholine was tested for the first time
19 days after right sympathectomy and 4 days after denervation on the left. There were no control observations before the operation. Almost daily tests were
performed during the first month of observation, throughout which time the TABLE 5 Summary of results insympathectomized male hypertensives CASE NO. 1
2
34
64
3
4
264
48
5
6
Earliest observed diminished response after sympathectomy in hours Consistently negative or very slight responses (days after sympathectomy) RIght
14
27
2 Left 62 Duration of observation period after 64 mo. 8 mo. sympathectomy
18
25
12
8 mo.
14 mo.
8
54 mo.
10 da.
sweat responses were usually diminished or absent, though not consistently so.
During the 2nd month of observation the response to acetyicholine became consistently strong on the left arm and the patient also noted the return of sweating caused by heat in this area. The latter observation was confirmed by a heat sweating test which indicated the return of nerve function in this area with consequent ability to perspire upon exposure to ordinary heat. Subsequent observations showed diminished responses. Thus, oscillations in the response of the sweat glands were noted over an 11 month period following sympathectomy. In some of the experiments, when a weak or negative response to acetyicholine
resulted, the test site was cleansed with alcohol and the iodine-starch mixture reapplied over the injection wheal. The second application, which was usually made 5 to 10 minutes after the injection, frequently was followed by a slightly stronger sweating response than was noted with the original test. No explanation is offered for this finding.
437
SWEAT RESPONSE TO ACETYLCHOLINE
3. Female patients with hypertension. Fifty-one tests were performed on four
patients. In these women, the diminished sweat responses to acetyicholine after sympathectomy were similar to those in men. However, the control areas TABLE 6 Man with scleroderma LERT ARM
DATE
RIGHT ARM
6/23/41
Right symp. Left symp.
7/7 7/11
7/12 7/14 7/15 7/16 7/17 7/18 7/19 7/21 7/23 7/24 7/25 7/26 7/28 7/29 7/30 7/31
0
+ +
0
+++? +
++ 0 0
0
0 0 0 0
0 0
++++ 0 ++ ++++
+
++++ 0 0
0 0
++
8/7 8/11 8/21
0 0 0
9/4
+
+
8/4 8/5
10/2 5/19/42
THIGH (coNTRoL)
++ ++++ +++ +++
+ +
+
++ ++++ ++ ++ ++ + ++++ +++ +++ ++++
TABLE 7 Local sweat response to intradermal acetyicholine (1:1000) in 100 control subjects
Men Women
NEGATIVE TO SLIGHT RESPONSE
NUMBER OP SUBJECTS
MODERATE TO VERY STRONG RESPONSE
59 41
56 (94.9%)
3 ( 5.1%)
4 ( 9.8%)
37 (90.2%)
usually gave relatively weak responses also. Therefore, no detailed data are presented on these cases.
4. Control subjects. Because of the unexpected difference in the sweat response noted in men as compared with women in normal areas of skin before sympathectomy, 152 tests were performed in 100 adult control subjects. Table 7 records the sweat responses of 59 men and 41 women.
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THE JOURNAL OF WVESTIGATIVE DERMATOLOGY
In 55 men tested with acetyicholine and 4 tested with mecholyl, the sweat response was moderate, strong or very strong in 56 instances (94.9%); in only 3 cases (5.1%) was there absent or very slight sweating. In 40 women injected with acetyicholine and one tested with mecholyl, the • sweat response was moderate to very strong in only 4 cases (9.8%); a negative or slight response was present in 37 subjects (90.2%). In some cases the tests were repeated after a varying number of weeks. Of ten controls, three men, whose initial response was weak, subsequently showed moderate or strong reactions. Because of this sex difference in response, the possibility of hormonal influences presented itself. Hence, a small group of children below the age of puberty were tested. In 4 boys ranging in age from 3 to 11 years, the sweat response was strong in 2 cases and negative in 2 cases. Four girls, 5 to 9 years of age, showed negative or very slight responses in 2 cases, and moderate or strong reactions in two. Many of the subjects for whom only acetyicholine responses are recorded in Table 7 were also tested with mecholyl. 43 tests were made in 39 subjects.
Parallel injections of the two drugs gave similar results. In some cases the mecholyl response was slightly greater than that with acetyicholine, but this was
not a constant finding. In 3 cases 1:1000 pilocarpine hydrochloride was injected with comparable results. Sweat responses in cats
Procedure. The sweat response to acetyicholine was also studied in the eat's paw in 3 animals. The appearance of sweat droplets was observed through a magnifying glass. Denervation of the sweat glands of the footpads was achieved
by section of the sciatic nerve. For each sweat test, 0.1 to 0.2 cc. of acetylcholine was injected subcutaneously into footpads of cats under ether anesthesia.
In each of the three cats the sweat response on the denervated side was first observed to become persistently negative or weak in 1, 5, and 13 days respectively, as compared with strong responses on the intact side. In one animal, the denervated paw gave a slightly stronger response than the normal paw for the first 24 hours, after which the reaction of the operated side became progressively weaker. The use of 1:200, 1:1000 or 1:5000 dilutions gave es-
sentially similar results. After sciatic nerve section, pallor was noted in the toe pads of the operated side. When a weakened and delayed sweat response after acetyicholine was obtained, the delay appeared to correspond to the time required for the pale toe pad to become reddened after injection. To test the assumption that the diminished sweat response to acetyicholine
after denervation might reflect a decrease in permeability of the sweat gland cell, the technique of vital staining was employed. Neutral red was found to be a suitable selective stain for sweat g'ands. The following mithod was used: The cat was anaesthetized with ether. 0.5 cc. of 0.5% aqueous solution of neutral red was injected into the footpad of the cat's paw. The footpad was excised 8 minutes after injection and placed in
SWEAT RESPONSE TO ACETYLCIIOLINE
439
formaldehyde. Frozen sections, 10 micra in thickness, were prepared, alcohol being used in the process. The tissue was mounted in glycerine. Histologic examination of the footpads of four cats failed to reveal any consistent difference in the degree of staining of denervated sweat glands as compared with intact glands. In general, the results were too variable, the technique too uncertain, and the number of animals too small to draw any definite conclusions at present regarding a change in permeability of the sweat gland cells after denervation. Further experimental work is in progress. DISCUSSION
1. Diminution in the sweat response to acetyicholine after sympathectomy
Investigators have indicated that the innervation of the sweat glands, although anatomically sympathetic, is pharmacologically parasympathetic. This is explained by the concept of two sets of postganglionic fibers in the thoracolumbar (sympathetic) system, adrenergic and cholinergic. 1) On stimulation of adrenergic fibers, at their endings sympathin, an epinephrine-like principle is liberated. The effect of adrenergic nerve impulse is identical with the effect of injection of epinephrine. Both effects are negated by ergotamine. 2) The cholinergic fibers liberate acetylcholine at their endings on stimulation. The effector cells are stimulated also by acetylcholine, pilocarpine, physostigmine,
and other parasympathetic drugs, and paralyzed by atropine. The fibers supplying the sweat glands are cholinergic although anatomically they course with the sympathetic chain, which otherwise contains adrenergic fibers only. In its direct action on sweat glands, acetylcholine conforms with the concept of a chemical mediator. According to the neurohumoral theory, the chemical mediator is liberated at the nerve ending and acts directly upon the effector organ. According to this theory, if the chemical mediator acetyicholine were injected directly into the gland-bearing area, a local sweating response would be expected also after denervation. Instead, our experimental results indicate that the sweating response becomes weak or absent after nerve section. Similar paradoxical behavior has been described for the parasympathomimetic drug, pilocarpine. There has been much controversy regarding the action of pilocarpine on sweat glands because many investigators have reported the disappearance of the sweat response to pilocarpine after denervation. Physiologists on the basis of Langley's (16) and Burn's (2) experiments hold that pilocarpine acts directly on the sweat glands, independent of the presence of a nerve supply. Clinical neurologists (20) question this concept and have advanced various theories of central and peripheral nervous action of the drug. Recent evidence (12) which attempts to explain this confusion indicates that the pilocarpine sweat response will disappear only after complete degeneration of postganglionic neurons supplying the sweat gland. However, the possibility of a direct action on the sweat gland cell by high concentrations of pilocarpine is admitted. In any case, it has frequently been observed that if sufficient time has elapsed after denervation, the sweat response to injected pilocarpine disappears.
440
THE JOURNAL OF INVESflGATIVE DERMATOLOGY
The weak or negative responses to acetyicholine of the denervated sweat glands represent an exception to the general rule of sensitization, as set forth by Cannon (3). As a general rule, smooth muscle and glands that are innervated by adrenergic and cholinergic nerve fibers give a greater response when chemically stimulated after denervation, especially if the chemical mediator, epinephrine or acetyleholine, supplies the stimulus. This has been explained by Cannon as a sensitization phenomenon, in which an increased irritability to chemical agents develops in the structure. Applying this principle to smooth muscle, Shen and Cannon (30) found increased sensitivity of the denervated iris to acetylcholine as compared with the normal iris. Simeone and Maes (32) noted similar results in submaxillary glands, to which the sympathetic nerves had been severed. However, when the parasympathetic innervation to the submaxillary gland was removed by severing the chorda tympani (mainly a preganglionic denervation), the expected increased salivary response to acetylcholine did not develop when tested two months after denervation, as noted by Pierce and Gregersen (29). Instead the drug apparently exerted a depressant influence, a result at variance with the concept of sensitization. Pilocarpine caused an increased response in this experiment. A second exception to Cannon's rule is represented by our experiments, in which the denervated sweat gland showed a decreased or absent response to acetylcholine instead of the increase to be expected were the structure "sensiti-
zed." It might be anticipated that acetyicholine would cause an increased response in the sweat gland after denervation. Instead, the response was diminished or absent after sympathectomy in our patients. In the experiments on cats, one animal showed a transitory increased response after denervation, followed soon by a decrease. In man, diminished sweat responses to intradermal acetylcholine were observed as early as 3 hours after sympathectomy in case 1. and 6 hours postoperatively in case 2. After a period of oscillation varying from 2 to 62 days, these responses became consistently negative or very slight and remained so during the entire period of observation—in some cases 6 to 8 months.
In all tests a concentration of the drug strong enough to paralyze its reflex action was used in order that the direct action of acetylcholine could be studied. Thus the diminished sweat response after sympathectomy is not to be explained on the basis of denervation of the reflex arc.
That sweating produced by intradermal acetyicholine is not due to simple mechanical squeezing out of sweat droplets from the gland by the pressure due to the volume of the injected drug is shown by experiments with saline controls. Langley (16) and Langely and Uyeno (17) injected Ringer's solution and ob-
tained a slight transient sweat secretion in the cat's footpad. This response
could be easily differentiated from the more profuse and more lasting response to pilocarpine. Similarly Coon and Rothman (4) noted slight or absent sweating with saline controls as compared to strong responses to acetyicholine. When
present, the reaction to saline consisted of sweat droplets limited to a small
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part of the footpad, whereas the response to the same volume of acetylcholine solution generally involved the whole surface of the injected toe pad. It might be argued that the concentration of the drug (1:1000) was not at the optimum level for direct stimulation of the sweat gland cells. This possibility is suggested by the observation of Myerson and co-workers (27) who noted that a greater concentration of mecholyl, when injected intradermally, is needed in fall than in summer to produce a good sweat response. This objection is answered by the fact that the control area gave strong responses to this concentration. In addition, some tests were repeated using a stronger concentration (1:200) of the drug with the same results. Acetyicholine is an unstable preparation, being destroyed rather rapidly by
the cholinesterase present in the tissues. To check the possibility that the weak or negative sweat responses might be due to this factor, parallel tests were carried out with mecholyl (acetyl-beta-methyicholine), a relatively stable derivative of acetylcholine, since it is only slightly hydrolyzed by cholinesterase. The
results with mecholyl were comparable to those with acetylcholine. In some cases, the response to mecholyl was slightly greater than that to acetylcholine, but not constantly. In view of the weak or negative reactions to acetylcholine after sympathectomy, it might be assumed that degneration of the sweat gland occurs relatively soon after denervation. However, the rapidity of disappearance of the sweat response would, in itself, militate against such a supposition. Furthermore, when we consider that after the development of a negative sweat response, oscil-
lating reactions are subsequently observed, it is evident that the denervated sweat glands are still functioning. That this early degeneration does not occur is attested by the following evidence: 1) Histologic study of skin obtained both before and after denervation of an area fails to show degeneration of the sweat glands following sympathectomy, according to a recent report (7). 2) Excessive local heating of a denervated area of skin after sympathectomy will evoke sweat-
ing in this region, even though ordinary heat sweating is not observed. The direct local response of the sweat glands to excessive heat was observed by Kuno (14) in cats 3—6 weeks after sciatic nerve section. 3) A type of response desig-
nated as "contact sweating" has been observed up to a year and a half after postganglionic sympathectomy. For some unexplained reason, when two sympathectomized areas of skin are placed in contact in a warm atmosphere, the corresponding sweat glands will secrete, according to Hyndman and Wolkiri (11). However, these authors do not distinguish sweating from insensible perspiration, which occur in such areas independent of sweat gland function (10). Thus the problem remains unsolved. We may explain the failure of a denervated area to sweat upon exposure to heat by the fact that heat sweating results
from a nervous impulse. Thus, after sympathectomy, there is no impulse to liberate acetyicholine at the nerve ending and as a result there is no activation of the sweat gland. However, when the "chemical mediator" is supplied by intradermally injected acetyicholine which suffuses the sweat gland-containing area with the drug, it is difficult to explain why a weak or negative reaction
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obtains. We cannot conclude that acetylcholine does not have a direct action on sweat glands because the sweat responses are sometimes positive, even though
slight, for long periods after sympathectomy, during which time variable responses are noted. Furthermore, by producing acetylcholine sweating in a locally anaesthetized area of skin in a normal control subject, we have additional evidence that the drug acts directly on the sweat gland. A possible explanation of the diminished or absent response is that alterations in the sweat gland cell-membrane, perhaps in the nature of a decreased perme-
ability, may occur after sympathectomy, thereby inhibiting the penetration of injected acetylcholine into the cell. This possibility was tested by the technique of vital staining with neutral red, which proved to be a good selective stain for sweat glands. However, the results were inconsistent.
Thus it appears that the response to acetylcholine of the denervated sweat gland is an exception to the basic law of sensitization of the structure after denervation. 2. Sex difference in the sweat response to acetyicholine of control subjects
The tendency to a stronger sweating response of men as compared with women after intradermal injection of acetyicholine has not been recorded previouslyas far as can be ascertained. No explanation is offered for this phenomenon, although many factors may be considered. Hormonal influences seem likely to play a role in causing this sex difference in response. It may or may not be significant that two of the four women who gave a stronger response than the others were pregnant. This sex difference in response was not due to atmospheric variations, since both sexes were tested under identical conditions of temperature and humidity.
The number of sweat glands per unit area of skin may play a role in the variations noted. It has been observed that relatively more sweat glands are present on the extremities of people whose costume leads to exposure of these areas (14). Infants have relatively more sweat glands per unit area of skin than adults. The number of children tested with acetylcholine was too small for the results to be conclusive. The relative amount of adipose tissue present in an area may influence the density of sweat gland distribution. But the acetylcholine sweat response did not appear to be related to the local or general fat ditribution. Evidence indicates that the amount of sweat does not depend entirely on the anatomic density of sweat glands in an area, since maniy of the
glands may not be in a functional state, as pointed out by Jurgensen (13). According to Adachi (1) there is no marked difference between children and adults with regard to the amOunt of sweating per unit of body surface. Although all sweat glands are present at birth, all do not function.
In any case it is not a likely assumption that the sweat glands in women could be so sparsely distributed in the area tested as to fail to give a visible response when directly stimulated by the drug in sufficiently high concentration. Furthermore, definite heat sweating was present in the same areas that responded poorly or negatively to acetycholine, as evidenced frequently by history, direct observation, or the starch-iodine visualization method.
SWEAT RESPONSE TO ACETYLCHOLINE
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There was no apparent correlation of heat sweating or emotional sweating with the sweat response to acetylcholine in the control subjects. For example, women usually had a poor response to the acetyleholine test, whether they perspired slightly or profusely in hot weather or in response to emotions. A preference for hot or cold weather bore no relationship to the sweat responsetested. CONCLUSIONS:
1. The local sweat response to intradermally injected acetylcholine diminishes or disappears after sympathectomy. 2. This finding is an exception to the rule of sensitization of structures after denervation (Cannon).
3. The local sweat response to the intradermal injection of acetyicholine is greater in men than in women.
We are greatly indebted to Dr. K. S. Grimson for his permission to make skin tests on his sympathectomized patients. Dr. E. M. K. Geiling placed the equipment of the Department of Pharmacology at our disposal and gave us valuable advice. Dr. J. M. Coon helped us greatly in the animal experiments. BIBLIOGRAPHY 1. ADACHI, J., Cited by KUNO, Y.: Functional variations in the human sweat glands. Jap. J. M. Sc., III, Biophysics, 5: 4*_5* (March) 1938. 2. BURN, J. H.: The relation of nerve supply and blood flow to sweating produced by pilocarpine. J. Physiol., 56: 232 (May) 1922. 3. CANNON, W. B.: A law of denervation. Am. J. Med. Sci., 198:737 (Dec.) 1939. 4. Cooar, J. M., AND ROTHMAN, S.: Sweat response to drugs with nicotine-like action. J.
of Pharm. and Exper. Therap.,73: 1 (Sept.) 1941. 5. DALE, H. H., AND FELDBERG, W.: The chemical transmission of secretory impulses to
the sweat glands of the cat. J. Physiol., 82: 121, 1934. 6. ELLIOTT, T. R. : Action of adrenalin. J. Physiol., 32: 401 (July) 1905. 7. GURNEY, R., AND BUNNELL, I. L.: A study of the reflex mechanism of sweating in the
human being; effect of anesthesia and sympathectomy. J. Clin. Invest., 21: 269 (May) 1942. 8. GUTTr,SAN, L., AND LIST, C. F.: Zur Topik und Pathophysiologie der Schweiss-Sekretion. Ztschr. f. d. ges. Neurol. u. Psychiat., 116: 504, 1928.
9. HAMPEL, C. W.: Effect of denervation on sensitivity to adrenine of smooth muscle in nictitating membrane of cat. Am. J. Physiol., 111: 611 (April) 1935. 10. HARE, K., AND HINSEY, J. C. : The autonomic nervous system. Ann. Rev. Physiol., 4: 434, 1942. 11. HYNDMAN, 0. R., AND WOLKIN, J.: Sweat mechanism in man: study of distribution of
sweat fibers from sympathetic ganglia, spinal roots, spinal cord and common carotid artery. Arch. Neurol. & Psychiat., 45: 446 (March) 1941. 12. HYNDMAN, 0. R., AND WOLKIN, J.: The pilocarpine sweating test. I. A valid indicator
in differentiation of preganglionic and postganglionic sympathectomy. Arch. Neurol. & Psychiat., 45: 992 (June) 1941. 13. JtJRqENSEN, E.: Mikrobeobachtungen der Schweiss-Sekretion der Haut des Mensehen unter Kontrastfarbung. Dtsch. Arch. kim. Med., 144: 193 (June) 1924. 14. KUNO, Y.: The Physiology of Human Perspiration. London, J. & A. Churchill, Ltd., 1934.
15. LANGLEY, J. N.: Observations on the physiological action of extracts of the supra-renal
bodies. J. Physiol., 27: 237 (Oct.) 1901.
444
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
16. LANGLEY, J. N.: The secretion of sweat. I. Supposed inhibitory nerve fibers in the posterior nerve roots; secretion after denervation. J. Physiol., 56: 110 (Feb.) 1922. 17. LANGLEY, J. N., AND [JYENO, K.: Secretion of sweat; effect of vaso-constriction and of
adrenaline. J. Physiol., 66: 206 (May) 1922. 18. LIST, C. F., AND PERT, M. M.: Sweat secretion in man. I. Sweating Responses in normal persons. Arch. Neurol. & Psychiat., 39: 1228 (June) 1938. 19. LIsT, C. F., AND PEEr, M. M.: Sweat secretion in man. II. Anatomic distribution of
sweating disturbances associated with lesions of the sympathetic nervous system. Arch. Neurol. & Psychiat., 40: 27 (July) 1938. 20. Lrsr, C. F., AND PEET, M. M.: Sweat secretion in man. III. Clinical observations on sweating produced by pilocarpine and mecholyl. Arch. Neurol. & Psyohiat., 40: 269 (Aug.) 1938.
21. Ltrco, J. V.: Sensitization of inhibited structures by denervation. Am. J. Physiol., 120: 179
(Sept.) 1937.
22. MARs, J. P.: Effect of removal of superior cervical ganglion on lachrymal secretion. Am. J. Physiol., 123: 359 (Aug.) 1938. 23.
MARRAZZI, R., AND MAREAZZI, A. S.: Ionic permeability of the sensitized nictitating
membrane of the cat. Am. J. Physiol., 133: 377, 1941. 24. MELTzER, S. J., AND AURE, C. M. : Studies on the "paradoxical" pupil-dilatationcaused
by adrenalin. Am. J. Physiol., 11: 28,37 (April) 1904. 25. MENG, C. W. : Role of cholinesterase in sensitization of muscle to acetyloholine. Chinese J. Physiol., 15: 143 (April 30) 1940. 26. MINOR, V.: Eines neues Verfahren zu der klinischen Untersuchung der Schweissabsonderung. Deutsche Ztsohr. f. Nervenh., 101: 302—308, 1927. 27. MYERSON, A., LOMAN, J., AND RIN1EL, M.: Human autonomic pharmacology; general
and local sweating produced by acetyl-beta-methyl-choline chloride (mecholyl). Am. J. M. Sc., 194: 75 (July) 1937. 28. PIERcE, F. R., AND GREGERSEN, M. I.: Changes in submaxillary secretory response to
pilocarpine after
section of chorda tympani. Am. J. Physiol., 120: 246 (Oct.) 1937.
29. ROTRMAN, S., AND CooN, 30.
31.
J. M.:
Axon reflex responses to acetyleholine
Invest. Dermat., 3: 79 (April) 1940. SEEN, S. C., AND CANNON, W. B.: Sensitization
of denervated
in the skin. J.
pupillary sphincter to
acetylcholine. Chinese J. Physiol., 10: 359 (May 15) 1936. SIMEONE, F. A.: Effect of regeneration of nerve supply on sensitivity of denervated nictitating membrane to adrenine. Am. J. Physiol.,120: 466, 1937.
32. SIMEONE, F. A., AND MARS, J. P.: Sensitization of submaxillary gland by sympathetic denervation. Am. J. Physiol., 125: 674 (April) 1939. 33. SOLLMANN, T.: A Manual of Pharmacology. Philadelphia, W. B. Saunders Company, 1942.
34. YOUMANS, W. B.: Similarity of effects of adrenalin and inhibitory sympathin on intestinal motility; sensitization by denervation. Am. J. Physiol., 123:424 (Aug.) 1938.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
94
linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
24. Wynn, C. H. and Iqbal, M.: Isolation of rat
skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.
human epidermis, p. 511, The Epidermis
Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-
No warranty is given about the accuracy of the copy.
Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.
Cell Biol., 27: 603, 1965.
27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-
C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.
30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.
31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-
lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.
32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.
33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.
34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:
Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.
43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.
44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.
45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.
46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,
Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by
Res., 33: 176, 1964.