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Regulation of human skin collagenase activity by hydrocortisone and dexamethasone in organ culture
Vol. 61, No. 3, 1974
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REGULATION OF HUMAN SKIN COLLAGENASE ACTIVITY BY HYDROCORTISONE AND DEXAMETHASONE IN ORGAN CULTURE Thomas J. Koob,
John J.
Jeffrey
and Arthur
2. Eisen
Division of Dermatology, Department of Medicine and the Department of Biological Chemistry Washington University School of Medicine St. Louis, Missouri 643110 Received
October
29,1974
SUMMARY Hydrocortisone and dexamethasone (90(-fluoro, 160(-methyl prednisolone) prevent the appearance of collagenase in cultures of normal human skin, human rheumatoid s novium and rat uterus. Hydrocortisone is maximally inhibiting at lo- v M and dexamethasone at lo-gM in culture medium. Neither steroid is an inhibitor of enzyme activity. The loss of collagenase activity in cultured tissue is not accompanied by detectable inhibition of protein synthesis. Reduction of enzyme activity in culture medium is concomitant with a parallel cessation of tissue collagen degradation, indicating that the tissue fails to produce active collagenase in the presence of physiologic levels of glucocorticoids. INTRODUCTION which
is
required
physiologic malian
Human skin for
control
collagen
intensive
the
found
catabolism
investigation
whether We report potent
is
could
affect
synthetic
analogue,
in vitro -___
catabolism
and thus
to exert
collagenase (1).
Normal
the control and is
of systems it
of mam-
currently
Normal
production of active
for
organ
human skin culture
(S).
metabolism
under
of human skin collagenase collagen was obtained
as previously
on the meta-
In view
of the pro-
to determine in human skin.
hydrocortisone
16 alpha-methyl
of the endogenous
AND METHODS
effects
was 'of interest
collagen
9 alpha-fluoro,
the appearance
degradation
major
of two glucocorticoids,
steroids
prepared
a specific
understood,
in vivo,
on the --in vitro
immediately
poorly
reported
(dexamethasone),
MATERIALS
synthesis
only
hormones
the effects
the --in vitro
of collagen
in mammals in a number
here
prevent
produces
(2-7).
of these
steroids
initiation
have been
of protein effects
culture,
of collagenase
Corticosteroids bolism
in organ
and its
prednisolone collagenase.
and concomitantly of cultured at surgery described
skin. and was (1).
Both inhibit
Vol.
61, No.
3, 1974
Briefly,
tissue
glucose
(Grand
BIOCHEMICAL
was cultured Island
02 and 5% CO2. lected
ethanol
(10 ul/lOO
desired
concentrations.
Collagenase native,
activity
14C-glycine
Tissue
collagen
determined soluble
from
crude
labeled
degradation
culture
to 0.05
M.
Penicillin
stock
fibrils
were
solutions directly
added
in
to obtain to the medium.
on reconstituted,
as previously
in culture
of 95%
medium was col-
Steroids
was added
was quantitated peptides
200 units/ml
medium was assayed
collagen
Medium-High
and each day's
appropriate
Cycloheximide
in
droxyproline-containing measured
ml)
COMMUNICATIONS
at 37°C in an atmosphere
daily
pH 7.4
Eagles
containing
was incubated
Tris-HCl,
RESEARCH
Modified
Company),
The medium was changed with
absolute the
Biological
and buffered
BIOPHYSICAL
in Dulbecco's
The tissue
and Streptomycin.
AND
described
(9).
by determining
the level
of hy-
medium
Hydroxyproline
by the method
of Bergmann
and Loxley
by measuring
the amount
of 3H-leucine
The effect
of hydrocortisone
(4,5).
(10).
Protein
was
synthesis
incorporated
into
was
CC1 COO0 in3
protein.
RESULTS AND DISCUSSION cultures
of human skin
Tables
explants
is
compared
to that
and dexamethasone of cycloheximide
on in
1 and 2. Both
dexamethasone,
at lo-8M,
and hydrocortisone, Table
Effect
of Hydrocortisone Collagen Degradation
Culture Conditions
Medium Collagenase
% Inhibition
Activity Cultures
and
Medium Hydroxyproline?
% Inhibition
115
4.05
Control
enzyme
1
on Collagenase in Human Skin *
at 10W7M, reduce
10W7M
0.39
90
38
68
II
10-8M
1.58
61
85
26
!I
lo-9M
4.02
0
120
0
Hydrocortisone,
Organ cultures of human skin were prepared as described in Methods. Aliquots (150 ul) of crude culture medium on the third day of culture were incubated for 18 hrs. with 200 ug native 14C-glycine labeled collagen fibrils (3600 cpm). *Results are expressed as ug collagen degraded per mg medium protein. -pedium hydroxyproline represents the total imino acid in the medium from two culture flasks (30 ml).
1084
Vol. 61, No. 3, 1974
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table Culture Conditions
Medium Collagenase*
Control
2 Medium Hydroxyproline+
% Inhibition
10.49
Dexamethasone,
% Inhibition
290
lo-*M
1.51
86
76
74
II
lo-9M
2.30
78
96
67
I,
lo-1°M
4.89
54
222
23
0
lo-1lM
6.26
41
179
38
II
lo-12M
9.10
14
298
0
II
lo-13M
11.20
0
319
0
0.53
95
Cycloheximide, 40 ug/ml
Cultures and assays were performed as described in Methods and in Table 1. *Collagenase activity is expressed as ug collagen degraded per mg medium protein. +Medium hydroxyproline represents the total imino acid in the medium from two culture flasks (30 ml).
activity ted,
by GO-90%.
inhibits
protein
Cycloheximide (Table
2),
inhibition
also
prevents
and the
reduction
However, 3),
medium,
hence
appear
neither
occurring
Concomitant
inhibit
culture
in the level
culture
(Tables
overall
the production
medium,
is
with
consistently
of the
mammalian protein
tissues
synthesis
at 10W7M in
culture
collagenase.
on steroid detectable
concentration inhibition
and 10W8M hydrocortisone.
inhibition
by these
steroids
of collagenase
of hydroxyproline-containing
1 and 2).
3).
the cultures
of active
dependent
(Table
result
as in other
affects
activity
dexamethasone, the
this
in
a direct
at 10 -%I and hydrocortisone
of collagenase
with
in
is
as expec-
by 95%-100%
of collagenase
of the steroids
is a reduction medium
synthesis
to selectively
at lo-12M
skin
in enzyme activity
dexamethasone
1 and 2) in the
of 40 ug/ml,
in the cultured
the appearance
protein
The inhibition (Tables
at a concentration
synthesis
of overall
(1,4,5). (Table
Cycloximide
It
has been
1085
shown
peptides that
the level
activity in the of these
Vol. 61, No. 3, 1974
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table Effect
3
of Glucocorticoids in Human Skin
on Protein Cultures
Synthesis
cpm 3H-leucine incorporated
% Inhibition
10,390
Control Dexamethasone
11,160
10 -8M
0
510
Cycloheximide Control Hydrocortisone
lo-'M
95
11,450
0
10,850
5
On the third Cultures were initiated and maintained as described in Methods. day of culture, 3H-leucine was added to culture medium (0.2 uc/ml) for 6 hrs. The tissue was harvested, blotted, weighed and homogenized in 5 volumes of 10% TCA and dissolved in 10 ml of Soluene (Nuclear-Chicago) at 6O"C, and radioacResults are expressed tivity determined by liquid scintillation spectrometry. as 3H-leucine cpm per g wet weight.
peptides
parallels
resorbing
tadpole
(manuscript
in a reduced
level
abolished.
--in vitro.
This
concentration
(Tables
maximally
of these
this
of collagen
true
in
production
the appearance
into
the
of active degradation
parallel
(4,5),
peptides.
catabolism
1 and 2) and is
is
in collagenase
peptides
collagen
uterus
laboratory
relationship
inhibit
the production endogenous
inhibition
in
any reduction
which
by inhibiting inhibit
this
of rat
of hydroxyproline-containing
the release
and hydrocortisone
studies
that
Thus,
concentrations
Thus,
in cultures
and recent
indicate
as well.
collagenase,
activity
(ll),
cultures
At steroid active
tailfin
in preparation)
human skin results
the collagenase
is with
culture
enzyme,
of medium is
dexamethasone
of human skin
dependent
upon steroid
the inhibition
of collagenase
activity. By two criteria measurement inhibit
of collagen
collagen
in human skin
then,
degradation,
degradation explants.
direct
assay it
of collagenase appears
by preventing The effect
appears
1086
that
in the medium glucocorticoids
the production to be highly
of active selective,
and can collagenase in that
Vol. 61, No. 3, 1974
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table Effect of Corticosteroids of Human Rheumatoid
4
on Collagenase Activity in Cultures Synovium and Involuting rat uterus Medium Collagenase*
Tissue Rat Uterus
Contrd
87
Dexamethasone Human Rheumatoid Synovium
10e7M
exhibit
is
no detectable An indication degradation
collagen
metabolism
skin,
totally
the
the --in
rheumatoid abolish
is
vitro
synovium,
cultures
enzyme activity (J 80%) is
The mechanism presently
Numerous corticoids ferose fibroblast phosphate
and rat
while
at 10
enzymes
can induce and alkaline
very
phosphatase
sane are added
is
induced
111 the systems to culture
which
on collagenase
and
the regulation other
collagenase
(Table
of 10V8M is
of
than
dexamethasone
human
similarly 4).
In
sufficient
to
uterus,
maximal
of rat
M. inhibit is known
col.lagenase about
of any eukaryotic
produced
of steroid
two tissues
in cultures
little
by cultured
the production
collagenase (13).
-I
in
uterus,
corticosteroids
in the regulation
specific
in
a concentration
reached
Indeed,
two steroids
that
and in per mg
synthesis.
significance
of active
95-lOO%,
by which
unknown.
corticosteroids
of these
the observation
production
synovial
inhibition
effect
95
by concentrations protein
may be of general
human rheumatoid
inhibits
on tissue
3 as described in Methods as ug collagen degraded
abolished
effect that
collagen
10maM
and assays performed activity is expressed
activity
83
63
Dexamethasone
collagenase
15
Control
Cultures were prepared Table 1. "Collagenase medium protein.
% Inhibition
of enzymes
(12).
A recent
by high described
have
here
role
report
shown
suggests
of
that
gluco-
amino that
trans-
cornea1
of dexamethasone
hydrocortisone well
is
process.
such as tyrosine
concentrations
medium at concentrations
1087
the primary
metabolic
cells
production
below
and dexamethathat
of the
BIOCHEMICAL
Vol. 61, No. 3, 1974
concentration lation
of hydrocortisone
of collagenase
observed
even at very
to an overall teroids
effect
of a physiologic metabolism
Rather,
observed. steroid
catabolism
investigation
role
is
and cannot Thus,
synthesis.
regulatory
in human skin
inhibition
concentrations
and collagen
ACKNOWLEDGMENTS This AM 12129
low
In no case has a stimu-
in human plasma.
on protein
on collagenase
indicative collagen
been
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of corticos-
we observe
of corticosteroids
was supported
be attributed
the effect
that
and possibly
consistently
other
animal
--in vitro
may be
on -~ in vivo tissues
by USPHS Grants
as well. HD 05291,
and AM 05611.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Eisen, A.Z., Jeffrey, J.J., and Gross, J., Biochim. Biophys. Acta -151:637 (1968). Bauer, E. A., Eisen, A.Z., Jeffrey, J.J., J. Invest. Dermatol. 55:359 (1970) Eisen, A.Z., Bauer, E.A., and Jeffrey, J.J., J. Invest. Dermator -59:50 (1972). Jeffrey, J.J., Coffey, R.J. and Eisen, A.Z., Biochim. Biophys. Acta (1971). -252:136 Jeffrey, J.J., Coffey, R.J., Eisen, A.Z., Biochim. Biophys. Acta -252:143 (1971). Jeffrey, J.J., and Koob, T.J., Endocrinology: Excerpta Medica Int'l Congr. Ser. 273:1115 (1973). Koob, T.J. andJeffrey, T.J., Biochim. Biophys. Acta 354:61 (1974). Ashmore, J. and Morgan, D. in __ The Adrenal A.Bxisenstein, ____ -)Cortex ed. Little, Brown, Boston, (1967), P. 249. Nagai, Y., Lapiere, G.M. and Gross, J. Biochem. 5:3123 (1966). Bergmann, I. and Loxley, R., Anal. Chem. 35:1961-(1963). Gross, J. and Bruschi, A.B., Dev. Biol. 26:36 (1971). Litwack, G. and Singer, S. in BiochemicaFActions of Hormones, G. Litwack, ed. Academic Press, New York (1972), p. 114. Hook, R.M., Hook, C.W. and Brown, S. I., Invest. bphthal. -12:771 (1973).
1088
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REGULATION OF HUMAN SKIN COLLAGENASE ACTIVITY BY HYDROCORTISONE AND DEXAMETHASONE IN ORGAN CULTURE Thomas J. Koob,
John J.
Jeffrey
and Arthur
2. Eisen
Division of Dermatology, Department of Medicine and the Department of Biological Chemistry Washington University School of Medicine St. Louis, Missouri 643110 Received
October
29,1974
SUMMARY Hydrocortisone and dexamethasone (90(-fluoro, 160(-methyl prednisolone) prevent the appearance of collagenase in cultures of normal human skin, human rheumatoid s novium and rat uterus. Hydrocortisone is maximally inhibiting at lo- v M and dexamethasone at lo-gM in culture medium. Neither steroid is an inhibitor of enzyme activity. The loss of collagenase activity in cultured tissue is not accompanied by detectable inhibition of protein synthesis. Reduction of enzyme activity in culture medium is concomitant with a parallel cessation of tissue collagen degradation, indicating that the tissue fails to produce active collagenase in the presence of physiologic levels of glucocorticoids. INTRODUCTION which
is
required
physiologic malian
Human skin for
control
collagen
intensive
the
found
catabolism
investigation
whether We report potent
is
could
affect
synthetic
analogue,
in vitro -___
catabolism
and thus
to exert
collagenase (1).
Normal
the control and is
of systems it
of mam-
currently
Normal
production of active
for
organ
human skin culture
(S).
metabolism
under
of human skin collagenase collagen was obtained
as previously
on the meta-
In view
of the pro-
to determine in human skin.
hydrocortisone
16 alpha-methyl
of the endogenous
AND METHODS
effects
was 'of interest
collagen
9 alpha-fluoro,
the appearance
degradation
major
of two glucocorticoids,
steroids
prepared
a specific
understood,
in vivo,
on the --in vitro
immediately
poorly
reported
(dexamethasone),
MATERIALS
synthesis
only
hormones
the effects
the --in vitro
of collagen
in mammals in a number
here
prevent
produces
(2-7).
of these
steroids
initiation
have been
of protein effects
culture,
of collagenase
Corticosteroids bolism
in organ
and its
prednisolone collagenase.
and concomitantly of cultured at surgery described
skin. and was (1).
Both inhibit
Vol.
61, No.
3, 1974
Briefly,
tissue
glucose
(Grand
BIOCHEMICAL
was cultured Island
02 and 5% CO2. lected
ethanol
(10 ul/lOO
desired
concentrations.
Collagenase native,
activity
14C-glycine
Tissue
collagen
determined soluble
from
crude
labeled
degradation
culture
to 0.05
M.
Penicillin
stock
fibrils
were
solutions directly
added
in
to obtain to the medium.
on reconstituted,
as previously
in culture
of 95%
medium was col-
Steroids
was added
was quantitated peptides
200 units/ml
medium was assayed
collagen
Medium-High
and each day's
appropriate
Cycloheximide
in
droxyproline-containing measured
ml)
COMMUNICATIONS
at 37°C in an atmosphere
daily
pH 7.4
Eagles
containing
was incubated
Tris-HCl,
RESEARCH
Modified
Company),
The medium was changed with
absolute the
Biological
and buffered
BIOPHYSICAL
in Dulbecco's
The tissue
and Streptomycin.
AND
described
(9).
by determining
the level
of hy-
medium
Hydroxyproline
by the method
of Bergmann
and Loxley
by measuring
the amount
of 3H-leucine
The effect
of hydrocortisone
(4,5).
(10).
Protein
was
synthesis
incorporated
into
was
CC1 COO0 in3
protein.
RESULTS AND DISCUSSION cultures
of human skin
Tables
explants
is
compared
to that
and dexamethasone of cycloheximide
on in
1 and 2. Both
dexamethasone,
at lo-8M,
and hydrocortisone, Table
Effect
of Hydrocortisone Collagen Degradation
Culture Conditions
Medium Collagenase
% Inhibition
Activity Cultures
and
Medium Hydroxyproline?
% Inhibition
115
4.05
Control
enzyme
1
on Collagenase in Human Skin *
at 10W7M, reduce
10W7M
0.39
90
38
68
II
10-8M
1.58
61
85
26
!I
lo-9M
4.02
0
120
0
Hydrocortisone,
Organ cultures of human skin were prepared as described in Methods. Aliquots (150 ul) of crude culture medium on the third day of culture were incubated for 18 hrs. with 200 ug native 14C-glycine labeled collagen fibrils (3600 cpm). *Results are expressed as ug collagen degraded per mg medium protein. -pedium hydroxyproline represents the total imino acid in the medium from two culture flasks (30 ml).
1084
Vol. 61, No. 3, 1974
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table Culture Conditions
Medium Collagenase*
Control
2 Medium Hydroxyproline+
% Inhibition
10.49
Dexamethasone,
% Inhibition
290
lo-*M
1.51
86
76
74
II
lo-9M
2.30
78
96
67
I,
lo-1°M
4.89
54
222
23
0
lo-1lM
6.26
41
179
38
II
lo-12M
9.10
14
298
0
II
lo-13M
11.20
0
319
0
0.53
95
Cycloheximide, 40 ug/ml
Cultures and assays were performed as described in Methods and in Table 1. *Collagenase activity is expressed as ug collagen degraded per mg medium protein. +Medium hydroxyproline represents the total imino acid in the medium from two culture flasks (30 ml).
activity ted,
by GO-90%.
inhibits
protein
Cycloheximide (Table
2),
inhibition
also
prevents
and the
reduction
However, 3),
medium,
hence
appear
neither
occurring
Concomitant
inhibit
culture
in the level
culture
(Tables
overall
the production
medium,
is
with
consistently
of the
mammalian protein
tissues
synthesis
at 10W7M in
culture
collagenase.
on steroid detectable
concentration inhibition
and 10W8M hydrocortisone.
inhibition
by these
steroids
of collagenase
of hydroxyproline-containing
1 and 2).
3).
the cultures
of active
dependent
(Table
result
as in other
affects
activity
dexamethasone, the
this
in
a direct
at 10 -%I and hydrocortisone
of collagenase
with
in
is
as expec-
by 95%-100%
of collagenase
of the steroids
is a reduction medium
synthesis
to selectively
at lo-12M
skin
in enzyme activity
dexamethasone
1 and 2) in the
of 40 ug/ml,
in the cultured
the appearance
protein
The inhibition (Tables
at a concentration
synthesis
of overall
(1,4,5). (Table
Cycloximide
It
has been
1085
shown
peptides that
the level
activity in the of these
Vol. 61, No. 3, 1974
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table Effect
3
of Glucocorticoids in Human Skin
on Protein Cultures
Synthesis
cpm 3H-leucine incorporated
% Inhibition
10,390
Control Dexamethasone
11,160
10 -8M
0
510
Cycloheximide Control Hydrocortisone
lo-'M
95
11,450
0
10,850
5
On the third Cultures were initiated and maintained as described in Methods. day of culture, 3H-leucine was added to culture medium (0.2 uc/ml) for 6 hrs. The tissue was harvested, blotted, weighed and homogenized in 5 volumes of 10% TCA and dissolved in 10 ml of Soluene (Nuclear-Chicago) at 6O"C, and radioacResults are expressed tivity determined by liquid scintillation spectrometry. as 3H-leucine cpm per g wet weight.
peptides
parallels
resorbing
tadpole
(manuscript
in a reduced
level
abolished.
--in vitro.
This
concentration
(Tables
maximally
of these
this
of collagen
true
in
production
the appearance
into
the
of active degradation
parallel
(4,5),
peptides.
catabolism
1 and 2) and is
is
in collagenase
peptides
collagen
uterus
laboratory
relationship
inhibit
the production endogenous
inhibition
in
any reduction
which
by inhibiting inhibit
this
of rat
of hydroxyproline-containing
the release
and hydrocortisone
studies
that
Thus,
concentrations
Thus,
in cultures
and recent
indicate
as well.
collagenase,
activity
(ll),
cultures
At steroid active
tailfin
in preparation)
human skin results
the collagenase
is with
culture
enzyme,
of medium is
dexamethasone
of human skin
dependent
upon steroid
the inhibition
of collagenase
activity. By two criteria measurement inhibit
of collagen
collagen
in human skin
then,
degradation,
degradation explants.
direct
assay it
of collagenase appears
by preventing The effect
appears
1086
that
in the medium glucocorticoids
the production to be highly
of active selective,
and can collagenase in that
Vol. 61, No. 3, 1974
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table Effect of Corticosteroids of Human Rheumatoid
4
on Collagenase Activity in Cultures Synovium and Involuting rat uterus Medium Collagenase*
Tissue Rat Uterus
Contrd
87
Dexamethasone Human Rheumatoid Synovium
10e7M
exhibit
is
no detectable An indication degradation
collagen
metabolism
skin,
totally
the
the --in
rheumatoid abolish
is
vitro
synovium,
cultures
enzyme activity (J 80%) is
The mechanism presently
Numerous corticoids ferose fibroblast phosphate
and rat
while
at 10
enzymes
can induce and alkaline
very
phosphatase
sane are added
is
induced
111 the systems to culture
which
on collagenase
and
the regulation other
collagenase
(Table
of 10V8M is
of
than
dexamethasone
human
similarly 4).
In
sufficient
to
uterus,
maximal
of rat
M. inhibit is known
col.lagenase about
of any eukaryotic
produced
of steroid
two tissues
in cultures
little
by cultured
the production
collagenase (13).
-I
in
uterus,
corticosteroids
in the regulation
specific
in
a concentration
reached
Indeed,
two steroids
that
and in per mg
synthesis.
significance
of active
95-lOO%,
by which
unknown.
corticosteroids
of these
the observation
production
synovial
inhibition
effect
95
by concentrations protein
may be of general
human rheumatoid
inhibits
on tissue
3 as described in Methods as ug collagen degraded
abolished
effect that
collagen
10maM
and assays performed activity is expressed
activity
83
63
Dexamethasone
collagenase
15
Control
Cultures were prepared Table 1. "Collagenase medium protein.
% Inhibition
of enzymes
(12).
A recent
by high described
have
here
role
report
shown
suggests
of
that
gluco-
amino that
trans-
cornea1
of dexamethasone
hydrocortisone well
is
process.
such as tyrosine
concentrations
medium at concentrations
1087
the primary
metabolic
cells
production
below
and dexamethathat
of the
BIOCHEMICAL
Vol. 61, No. 3, 1974
concentration lation
of hydrocortisone
of collagenase
observed
even at very
to an overall teroids
effect
of a physiologic metabolism
Rather,
observed. steroid
catabolism
investigation
role
is
and cannot Thus,
synthesis.
regulatory
in human skin
inhibition
concentrations
and collagen
ACKNOWLEDGMENTS This AM 12129
low
In no case has a stimu-
in human plasma.
on protein
on collagenase
indicative collagen
been
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of corticos-
we observe
of corticosteroids
was supported
be attributed
the effect
that
and possibly
consistently
other
animal
--in vitro
may be
on -~ in vivo tissues
by USPHS Grants
as well. HD 05291,
and AM 05611.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Eisen, A.Z., Jeffrey, J.J., and Gross, J., Biochim. Biophys. Acta -151:637 (1968). Bauer, E. A., Eisen, A.Z., Jeffrey, J.J., J. Invest. Dermatol. 55:359 (1970) Eisen, A.Z., Bauer, E.A., and Jeffrey, J.J., J. Invest. Dermator -59:50 (1972). Jeffrey, J.J., Coffey, R.J. and Eisen, A.Z., Biochim. Biophys. Acta (1971). -252:136 Jeffrey, J.J., Coffey, R.J., Eisen, A.Z., Biochim. Biophys. Acta -252:143 (1971). Jeffrey, J.J., and Koob, T.J., Endocrinology: Excerpta Medica Int'l Congr. Ser. 273:1115 (1973). Koob, T.J. andJeffrey, T.J., Biochim. Biophys. Acta 354:61 (1974). Ashmore, J. and Morgan, D. in __ The Adrenal A.Bxisenstein, ____ -)Cortex ed. Little, Brown, Boston, (1967), P. 249. Nagai, Y., Lapiere, G.M. and Gross, J. Biochem. 5:3123 (1966). Bergmann, I. and Loxley, R., Anal. Chem. 35:1961-(1963). Gross, J. and Bruschi, A.B., Dev. Biol. 26:36 (1971). Litwack, G. and Singer, S. in BiochemicaFActions of Hormones, G. Litwack, ed. Academic Press, New York (1972), p. 114. Hook, R.M., Hook, C.W. and Brown, S. I., Invest. bphthal. -12:771 (1973).
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