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Disruption
of
the
Golgi
Zone
and
Inhibition of
the
Conversion
of
Proparathyroid
Hormone
to
Parathyroid
Hormone
in
Human
Parathyroid
Tissue
by
Tris(hydroxymethyl)aminomethane
Douglas
H.
McGregor,
MD,
Luke
L.
H.
Chu,
PhD,
Ronal
R.
MacGregor,
PhD,
and
David
V.
Cohn,
PhD
Tris(hydroxymethyl)aminomethane
(Tris,
Tromethamine,
THAM)
and
other
non-
amphoteric
ammes
were
previously
reported
to
inhibit
the
conversion
of
pro-
parathyroid
hormone
to
parathyroid
hormone
in
bovine
parathyroid
glands
incubated
in
citro.
This
inhibition
correlated
with
a
striling
dilation
of
the
Golgi
complex.
This
work
has
now
been
extended
to
normal,
hyperplastic,
and
adenomatous
parathyroid
glands
from
human
subjects.
The
tissues
were
incubated
for
up
to
3
hours
with
3H-
leucine
in
physiologic
solutions
(control)
or
in
the
same
solutions
containing
50
mM
Tris.
In
one
case,
diethylamine
also
was
tested.
Electron
microscopy
revealed
that
the
amines
produced
a
dilation
of
the
Golgi
complex
and
swelling
of
vesicles,
pre-
dominantly
in
the
region
of
the
Golgi
zone.
Other
organelles
were
normal
in
appear-
ance.
During
the
same
period,
Tris
reduced
by
sixfold
the
ratio
of
parathvroid
hormone
to
proparathyroid
hormone,
from
a
control
value
of
2:
1
to
1:
3.
It
was
apparent
that
Tris
exerted
the
same
biochemical
and
morphologic
actions
in
human
parathvroid
tissues
as
it
was
previously
shown
to
do
in
bovine
glands.
These
studies
support
the
concept
that
the
Golgi
zone
is
that
region
in
the
parathyroid
gland
in
which
pro-
parathyroid
hormone
to
parathyroid
hormone
conversion
is
initiated
and
that
Tris
inhibits
this
conversion
through
disruption
of
the
converting
site.
(Am
J
Pathol
87:553-568,
1977)
IT
WAS
REPORTED
PREVIOUSLY
that
when
bovine
parathyroid
tissue
slices
were
incubated
in
physiologic
buffers
containing
Tris
or
other
nonamphoteric
amines
such
as
glycinamide
and
diethylamine,
there
oc-
curred
a
time-dependent
and
dose-dependent
swelling
of
the
Golgi
zone
of
the
chief
cells.1'2
The
swelling
was
paralleled
by
inhibition
of
para-
thvroid
hormone
formation
and
an
accumulation
of
its
precursor,
pro-
parathyroid
hormone.
This
action
of
Tris
required
the
intact
tissue,
since
the
amine
did
not
affect
the
conversion
of
proparathyroid
hormone
to
parathyroid
hormone
in
a
cell
homogenate.
Based
on
these
and
other
From
the
Calcium
Research
Laboratorv
and
the
Laboraton-
Service,
Veterans
Administration
Hospital,
Kansas
City.
M1issouri,
and
the
Unisersity
of
Kansas
School
of
Medicine.
Kansas
City.
Kansas.
Presented
in
part
at
the
Seventy-third
Annual
Meeting
of
the
American
Association
of
Pathologists
and
Bacteriologists,
Boston. Mass..
1arch
22.
1976.
Supported
in
part
by
Grant
AM-18.323
from
the
National
Institute
of
Arthritis.
Metabolism.
and
Digestise
Diseases.
National
Institutes
of
Health
Accepted
for
publication
February
9.
1977
Address
reprint
requests
to
Dr
Douglas
H.
McGregor,
Calcium
Research
Laboratory.
Veterans
Administration
Hospital,
4801
Linswood
Boulesard,
Kansas
City.
M10
64128.
553
554
McGREGOR
ET
AL.
American
Journal
of
Pathology
data,
it
was
postulated
that
Tris
blocked
the
conversion
of
proparathyroid
hormone
to
parathyroid
hormone
by
mechanically
disrupting
the
convert-
ing
site
of
the
Golgi
zone.3'6
Although
the
mechanism
by
which
Tris
and
the
other
amines
act
is
unknown,
it
was
suggested
1
that,
if
these
agents
produced
similar
struc-
tural
alterations
in
other
tissues
and
cells,
they
might
comprise
a
unique
series
of
specific
probes
for
the
study
of
structure-function
relationships
in
secretory
cells.
To
this
end,
it
seemed
worthwhile
to
first
establish
that
the
disruptive
action
of
Tris
on
the
parathyroid
Golgi
zones
was
not
confined
solely
to
the
bovine
gland.
We
report
herein
that,
in
normal,
hyperplastic,
and
adenomatous
parathyroid
glands
from
humans,
Tris
causes
extensive
vesicle
formation
that,
as
in
bovine
tissue,
appears
to
be
of
Golgi
origin
and
that
this
morphologic
change
correlates
with
the
inhibition
of
para-
thyroid
hormone
formation
from
proparathyroid
hormone.
Materials
and
Methods
Preparation
and
Incubation
of
Tissue
Slices
Fresh
normal
parathyroid
glands
were
obtained
at
autopsy
from
10
subjects
less
than
3
hours
postmortem.
Fresh
hyperplastic
or
adenomatous
parathyroid
tissue
from
6
subjects
was
obtained
less
than
15
minutes
after
removal
at
surgery.
Each
tissue
was
cut
into
2
mm
X
2
mm
X
1
mm
pieces
and
incubated
in
either
Earle's
balanced
salt
solution
(EB
solution),
Minimum
Essential
Medium-10%
fetal
calf
serum
(MEM
solution),
or
Hanks'
solution-5%
fetal
calf
serum
for
60
minutes
as
indicated.
The
tissues
were
then
divided
into
two
portions.
One
was
further
incubated
for
30
minutes
in
fresh
buffer
solution;
the
other
was
incubated
in
the
same
solution
with
the
addition
of
either
50
mM
Tris,
pH
7.4,
or
50
mM
diethylamine,
pH
7.4.
The
tissues
were
then
incubated
in
the
same
fresh
media
but
containing
3H-leucine
or
3H-lysine
for
2
to
3
additional
hours.
Portions
of
the
tissue
were
removed
at
appropriate
times
for
electron
microscopic
examination.
The
osmotic
strength
of
the
control
and
experimental
solutions
was
not
normally
equalized
since
we
found
earlier
that
these
small
differences
had
no
effect
on
either
the
morphology
or
measured
biochemical
activity
of
the
tissues.'
Isolation
of
Proparathormone
and
Parathormone
After
incubation
the
tissue
and
medium
were
homogenized
together
in
5
ml
of
8
M
urea-0.2
M
HC1-0.
1
M
cysteine
after
which
25
ml
of
a
similar
10%
homogenate
of
frozen
bovine
glands
were
added
to
serve
as
carrier
protein.
The
mixture
was
processed
as
described
previously
7
through
the
carboxymethylcellulose
(CM-cellulose)
chromatogra-
phy
step
in
order
to
separate
radioactive
proparathyroid
hormone
and
parathyroid
hor-
mone.
Aliquots
of
each
fraction
were
assayed
for
radioactivity
using
Phosphor
Solution
B.
The
recovery
of
the
radioactive
hormonal
peptides
was
followed
by
inclusion
of
bovine
'4C-proparathyroid
hormone
and
'4C-parathyroid
hormone
as
markers
in
the
initial
ho-
mogenate.
Recovery
was
about
50%
during
the
course
of
the
extraction
procedure
and
was
unaffected
by
the
presence
of
Tris
in
the
incubation
media.
The
recoveries
of
pro-
parathyroid
hormone
and
parathyroid
hormone
from
CM-cellulose
columns
averaged
55%
and
85%,
respectively,
and
the
values
of
the
prohormone
and
hormone,
unless
stated
otherwise,
were
not
adjusted
for
these
losses.
The
incorporation
of
3H-amino
acids
into
Vol.
87,
No.
3
TRIS
AND
HUMAN
PARATHYROID
555
June1977
total
proteins
of
the
tissue
was
measured
in
the
insoluble
1O0c
trichloroacetic
acid
precipi-
tate
derived
from
a
sample
of
the
original
tissue
homogenate.
The
precipitate
was
collected
bv
centrifugation,
washed
several
times
with
10%
trichloroacetic
acid,
extracted
with
5%c
hot
trichloroacetic
acid,
dissolved
in
a
tissue
solubilizer
(NCS,
Amersham/Searle)
and
assaved
for
radioactivity
using
Phosphor
Solution
A.
Radioactive
assay
was
conducted
by
liquid
scintillation
spectroscopy
using
either
5
g/liter
of
2,5-diphenvloxazole
(PPO)
and
0.12
gjliter
of
1,4-bis-2-(5-phenvloxazolvl)-
benzene
(POPOP)
in
toluene
(Phosphor
Solution
A)
or
3.33
g/liter
of
PPO
and
0.08
g/liter
of
POPOP
in
a
solution
of
toluene/Triton
X-100
(2:
1)
(Phosphor
Solution
B).
The
efficiency
of
counting
and
degree
of
quenching
for
samples
of
interest
were
determined
by
means
of
an
internal
standard,
either
14C-toluene,
sH-toluene,
or
'H2O.
Eectron
Mi
c
Exaroination
Fresh
tissue
and
tissues
after
incubation
were
fixed
in
4%
glutaraldehyde,
0.1
\
sodium
phosphate-buffered
at
pH
7.2,
postfixed
in
1.33%c
sym-collidine-buffered
osmium
tetrox-
ide
(pH
7.4),
dehydrated
in
ethanol,
and
embedded
in
Araldite-Epon.'
Thin
sections
were
cut
with
a
diamond
knife
on
an
LKB
ultramicrotome
and
were
stained
with
lead
citrate9
and
uranvl
acetate.10
Materials
3H-leucine
was
purchased
from
New
England
Nuclear
Corporation,
Boston,
NMass.;
Tris
from
Sigma
Chemical
Company,
St.
Louis,
Mto.;
and
carboxymethylcellulose
(CNM-52)
from
Reeve
Angel,
Clifton,
N.J.
Radioactive
bovine
'4C-proparathvroid
hormone
and
14C-
parathyroid
hormone
markers
were
biosynthesized
and
isolated
as
earlier
described."
All
other
chemicals
were
reagent
grade
and
w
ere
purchased
from
various
chemical
suppliers.
Results
Ultrastructural
studies
were
performed
on
the
human
parathyroid
tis-
sue
before
and
after
in
vitro
incubation.
The
results
with
four
representa-
tive
samples-a
normal
parathyroid
gland,
one
with
chief
cell
hyper-
plasia,
an
oxyphil
adenoma,
and
a
chief
cell
adenoma-will
now
be
described.
Figure
1
shows
the
ultrastructural
appearance
of
normal
parathyroid
tissue
removed
3
hours
after
death
and
before
incubation.
Except
for
mild
degenerative
changes
such
as
swollen
mitochondria,
the
cellular
struc-
tures-including
plasma
membrane,
nucleus,
endoplasmic
reticulum,
se-
cretorv
granules
and
Golgi
complex-appeared
normal.
Figure
2
shows
normal
parathyroid
tissue
following
incubation
for
2
hours
in
MEM
solution.
All
cellular
structures
including
the
mitochondria
appeared
nor-
mal.
Thus,
incubation
in
the
control
buffer
appeared
to
improve
the
morphologic
integrity
of
the
tissue.
The
tissue
maintained
this
normal
appearance
up
to
at
least
3
hours
of
incubation.
Identical
results
were
obtained
with
the
other
incubation
solutions.
Figure
3
shows
parathyroid
tissue after
incubation
for
1
hour
in
the
control
M
EM
solution
followed
by
1
hour
in
the
same
buffer
containing
50
mM
Tris.
The
cells
contained
556
McGREGOR
ET
AL
American
Journal
of
Pathology
multiple
membrane-limited
vacuoles
which
were
approximately
spherical
and
ranged
from
0.15
to
1.7
j,
in
diameter.
The
membranes
of
the
vacuoles
were
sometimes
incomplete
and
coalesced
with
adjacent
vacu-
oles.
Most
of
the
vacuoles
appeared
empty,
but
they
occasionally
con-
tained
distorted
membranous
material,
secretory
granule-like
material,
or
lipid-like
material.
The
vacuoles
appeared
to
be
mostly
concentrated
in
the
apparent
region
of
the
Golgi
apparatus.
Normal
Golgi
apparatuses
were
rarely
if
ever
identified
in
the
cells
of
any
of
the
tissue
that
were
incubated
in
Tris
solutions
for
periods
of
1
hour
or
more.
Furthermore,
secretory
granules
were
rarely
identified
in
these
cells.
In
contrast,
the
remainder
of
the
cell
organelles
presented
a
normal
appearance.
Figure
4
shows
the
tissue
after
incubation
for
1
hour
in
MEM
solution
followed
by
1
hour
in
the
same
buffer
containing
diethylamine.
Multiple
vesicles
were
seen
that
resembled
those
resulting
from
incubation
with
Tris
and
had
a
configuration
suggestive
of
Golgi
origin.
The
ultrastructural
appearance
of
parathyroid
tissue
with
primary
chief
cell
hyperplasia
after
removal
at
surgery
was
normal
except
for
occasional
mild
nuclear
disruption
and
mitochondrial
swelling.
After
incubation
of
a
sample
of
this
tissue
for
2
hours
in
EB
solution
(Figure
5),
it
was
somewhat
improved
in
appearance
over
the
unincubated
sample.
Following
in-
cubation
for
1
hour
in
EB
solution
containing
Tris,
however,
the
tissue
contained
multiple
vesicles;
and
normal
Golgi
complexes
were
not
ob-
served
(Figure
6).
All
other
cellular
structures
appeared
relatively
normal.
The
ultrastructural
appearance
of
a
functioning
oxyphil
adenoma
im-
mediately
after
removal
at
surgery
is
presented
in
Figure
7.
In
general,
all
cells-including
the
oxyphil
cells,
the
transitional
oxyphil
cells,
and
the
rare
chief
cells-were
morphologically
intact.
Likewise,
all
of
the
cellular
organelles
including
the
Golgi
complexes
appeared
to
be
normal.
After
2
hours
in
EB
solution,
the
tissue
continued
to
exhibit
a
normal
appearance.
When
a
sample
of
the
adenoma
was
incubated
in
EB
solution
containing
Tris,
multiple
vesicles
formed
(Figure
8),
and
there
was
an
absence
of
most
recognizable
Golgi
complexes.
Secretory
granules
were
rarely
identi-
fied.
All
other
structures
appeared
normal.
At
some
sites,
structures
inter-
mediate
between
normal
Golgi
vacuoles
and
the
fully
distended
vacuoles
were
seen.
The
ultrastructural
appearances
of
a
sample
of
a
chief
cell
adenoma
examined
immediately
after
removal
at
surgery
and
after
60
minutes
of
incubation
in
EB
solution
are
shown
in
Figures
9
and
10,
respectively.
The
cellular
structures
were
generally
intact.
Figures
11
and
12
show
the
tissue
incubated
for
30
and
60
minutes,
respectively,
in
EB
solution
containing
Tris.
At
30
minutes
there
appeared
to
be
mild
to
moderate
distension
of
Vol.
87,
No.
3
TRIS
AND
HUMAN
PARATHYROID
557
June
1977
Golgi
apparatuses
and
focal
vesicle
formation
which
was
more
prominent
at
60
minutes.
The
capacity
of
the
various
parathvroid
samples
to
synthesize
pro-
parathyroid
hormone
and
parathyroid
hormone
upon
incubation
in
the
control
and
Tris-containing
buffers
was
assessed.
Among
the
individual
experiments,
there
was
substantial
variability
in
incorporation
of
radio-
activity
into
total
protein.
Overall
parathyroid
hormone
and
pro-
parathyroid
hormone
represented
from
1
to
7%
of
the
total
acid-insoluble
protein.
The
appearance
of
CM-cellulose
chromatograms
from
two
typi-
cal
studies-normal
parathyroid
tissue
(Text-figure
1)
and
an
oxyphil
adenoma
(Text-figure
2)-are
shown.
After
2
to
3
hours
of
incubation
in
control
buffer,
the
majority
of
the
radioactivity
in
the
sample
was
in
parathyroid
hormone
and
proparathvroid
hormone,
with
about
twice
as
much
radioactivity
in
the
former
than
in
the
latter.
When
samples
of
the
tissues
were
incubated
in
the
Tris-containing
EB
solution,
the
amount
of
radioactivity
in
parathyroid
hormone
was
substantially
less
and
the
amount
of
proparathyroid
hormone
was
greater
by
about
the
same
amount.
The
radioactivity
in
the
other
(unidentified)
peaks
in
the
elution
profile
was
about
the
same.
In
the
total
of
16
normal,
hvperplastic,
and
10
PTH
CONTROL
+
TRIS
8
X103
E
>
~~~~~~~~~~PTH
U
.-
0ETFCtR
-abwmtslcluoeinecag
lto
prfi_
PofParTH-rihomn
ES
4~~~I
<c~
2
~~ProPTH
0
0
2
00
10
0
30
FRACTION
TEXT-FIGUIRE
I-Carbow~methy
1
cellulose
ion
exchange
elution
profiles
of
parathy
roid
hormone.
proparathyroid
hormone,
and
related
peptides
derised
from
a
normal
human
gland
after
incubation
in
control
or
Tris-containing
buffer.
Approximately
65
mg
of
tissue
w-ere
incubated
in
Earle's
balanced
salt
solution
w
ith
25
MCi
of
'H-ly
sine
for
3
hours
after
a
1-hour
preincubation
as
described
in
Materials
and
Methods.
Tris
was
added
to
the
experimental
buffer
at
a
concentration
of
50
mM1.
The
elution
positions
of
parathyroid
hormone
(PTH)
and
proparathyroid
hormone
(ProPTH)
are
indicated.
bbt
MCGHREO
EtI
AL.
American
Journal
of
Pathology
3
x10
3
CONTROL
+TRIS-
PTH
ProPTHf1
(
NTR
h{TEXT-FIGURE
2-Carboxy-
methyl
cellulose
ion
exchange
o-
E
ProPTH
Jf
elution
profiles
of
parathyroid
ProPTH
hormone,
proparathyroid
hor-
i t
I
mone,
and
related
peptides
de-
U
12
rived
from
a
human
parathyroid
oxv
phil
adenoma
after
incubation
va
l
l l
l
\
PTH
in
control
or
Tris-containing
buf-
-6C
1
-
I
l
I I
_
C
z
I
W
_
fer.
The
conditions
were
similar
<
X
1WF
t
\
\
|
'
s
to
those
described
in
the
legend
to
Text-figure
1
except
that
50
tCi
of
3H-leucine
was
used
and
1%%
_
s
X
the
buffer
vas
Hanks'
solu-
tion-57c
fetal
calf
serum.
0
10
20
30
40
0
10
20
30
40
FRACTION
adenomatous
parathyroid
glands,
the
average
amount
of
radioactive
para-
thyroid
hormone
as
a
percentage
of
the
total
hormonal
peptides
was
reduced
from
76
±
3%
(SD)
to
27
±
3%
(SD)
when
the
tissues
were
incubated
in
the
amine-containing
buffer.
Discussion
Our
data
show
that
Tris
(and
when
tested,
diethylamine)
produced
large
single
membrane-limited
vacuoles
when
normal,
hyperplastic,
and
adenomatous
human
parathyroid
tissues
were
incubated
in
solutions
con-
taining
this
amine.
In
contrast,
incubation
of
samples
of
the
same
tissue
in
the
physiologic
buffers
that
did
not
contain
Tris
yielded
normal
appearing
tissues.
Indeed,
the
tissues
incubated
in
the
control
solutions
appeared
structurally
as
good
or
better
than
did
the
unincubated
autopsy
speci-
mens.
The
amine-induced
vesicles
were
detected
in
smaller
dimensions
at
30
minutes
and
were
generally
fully
distended
by
1
hour
of
incubation.
The
impression
from
viewing
many
sections
similar
to
those
in
Figures
1-12
is
that
these
vesicles
represent
at
least
in
part
swollen
cisternae
and
vesicles
of
Golgi
origin.
This
impression
is
strengthened
by
the
almost
total
disappearance
of
recognizable
Golgi
organelles
in
the
cells.
The
uniqueness
of
the
morphologic
alteration
brought
on
by
Tris
is
empha-
sized
by
the
normal
appearance
of
the
other
major
cellular
organelles,
except
for
the
apparent
decrease
in
number
of
identifiable
secretory
granules.
Regarding
this
latter
finding,
it
is
felt
that
the
number
of
granules
and
extent
of
tissue
sampling
was
not
sufficient
to
quantitate
or
determine
statistical
significance
of
this
alteration.
The
morphologic
changes
were
accompanied
by
a
marked
accumula-
tion
of
newly-synthesized
proparathyroid
hormone
and
an
equivalent
i-IT,
At
Vol.
87,
No.
3
TRIS
AND
HUMAN
PARATHYROID
559
June
1977
lessening
in
the
amount
of
newly-synthesized
parathyroid
hormone
in
the
tissue,
in
accord
with
the
results
reported
earlier
with
bovine
tissue.1
Thus
the
present
data
support
the
previous
hypothesis
1
that
Tris
disrupts
the
Golgi
structure-the
site
of
conversion
of
proparathyroid
hormone
to
parathyroid
hormone3
`and
in
so
doing
inhibits
the
enzymatic
con-
version
of
the
prohormone
to
hormone.
It
will
be
of
interest
to
determine
if
other
compounds
such
as
the
antibiotic
ionophore
X537A,
reported
to
cause
swelling
of
the
Golgi
apparatus,
also
are
associated
with
inhibition
of
this
enzymatic
conversion.'
One
additional
point
of
interest
is
the
superficial
similarity
in
ultrastruc-
ture
between
human
primary
water-clear
cell
hyperplasia
13
and
the
para-
thvroid
tissue
of
the present
study
following
incubation
with
Tris.
Both
types
of
tissue
have
many
intracytoplasmic
spherical
membrane-limited
vacuoles.
The
mechanism
of
the
multiple
vesicle
formation
in
those
two
processes
appears
to
be
different,
however,
because
a)
in
contrast
to
our
Tris-incubated
tissue,
water-clear
cells
demonstrate
readily
identifiable
and
nondistended
Golgi
zones
and
b)
evidence
reported
elsewhere
14
suggests
that
inhibition
of
the
conversion
of
proparathyroid
hormone
to
parathyroid
hormone
is
not
a
feature
associated
with
the
vesicle
formation
of
water-clear
cell
hyperplasia
since
the
percentage
of
proparathyroid
hormone
[proparathyroid
hormone/(proparathyroid
hormone
+
para-
thvroid
hormone)]
in
tissue
from
1
case
of
clear
cell
hyperplasia
was
49%,
whereas
the
mean
percentage
of
proparathyroid
hormone
in
6
cases
of
adenoma
was
61
%
and
in
4
bovine
glands
was
50
%.
Our
findings
may
have
substantial
clinical
significance.
Tris
has
been
recommended
for
the
correction
of
metabolic
acidosis
associated
with
cardiac
bvpass surgery
and
cardiac
arrest
and
the
correction
of
acidity
of
acid-citrate-dextrose
(ACD)
blood
used
in
cardiac
bypass
surgerv
15
and
exchange
transfusion.16
It
would
appear
that
the
agent
has
direct
toxic
actions
possibly
induced
by
the
basicity
of
the
unneutralized
amine
15,17,18
or
due
to
its
metal-chelating
property.'9'"
These
actions,
however,
are
expressed
almost
immediately
and
may
not
be
related
to
the
Golgi
changes
we
observed.
On
the
other
hand,
in
longer
term
studies
(that
is,
those
extending
more
than
a
few
minutes),
Tris
may
exert
different
pharmacologic
effects.
For
example,
it
has
been
reported
to
cause
hepatic
injury
(possibly
unrelated
to
alkalinity
of
solutions)
when
infused
into
infants
via
the
umbilical
vein
to
combat
idiopathic
respiratorv
distress
svndrome.21
In
vitro,
Tris
is
known
to
be
toxic
to
cells
in
culture,
2
to
cause
release
of
specific
membrane-associated
proteins
from
Escherichia
coli,
suggesting
that
it
may
react
uniquely
with
cell
membranes,'
and
to
specifically
increase
secretion
of
insulin
by
pancreatic
islet
cells
in cul-
560
McGREGOR
ET
AL.
American
Journal
of
Pathology
ture.24
In
such
situations,
it
is
possible
that
the
action
of
Tris
might
be
related
to
a
disruption
of
the
Golgi
or
other
subcellular
organelles
in
cells
at
large.
Such
data
reinforce
the
need
to
investigate
more
closely
the
interaction
of
Tris
with
cells
and
organs
in
vitro
and
in
vivo.
As
referred
to
earlier,
one
benefit
of
such
a
study
might
be
to
learn
if
Tris
can
serve
as
a
specific
intracellular
probe
of
Golgi
function.
This
would
be
the
case
if
it
could
be
shown
that
it
produces
morphologic
alterations
in
organs
other
than
the
parathyroid
and
in
the
processing
of
other
peptide
molecules
(such
as
collagen,
insulin,
pancreatic
and
parotid
digestive
enzymes,
immunoglobulins,
and
albumin)
believed
to
follow
the
Golgi
route.
References
1.
Chu
LLH,
MacGregor
RR,
Hamilton
JW,
Cohn
DV:
Conversion
of
proparathyroid
hormone:
The
use
of
amines
as
specific
inhibitors.
Endocrinology
95:1431-1437,
1974
2.
Cohn
DV,
MacGregor
RR,
Chu
LLH,
Hamilton
JW:
Structure-function
relation-
ships
in
the
synthesis,
packaging
and
secretion
of
parathyroid
gland
hormones.
Calcium
Regulating
Hormones.
Edited
by
RV
Talmage,
M
Owen,
JA
Parsons.
New
York,
American
Elsevier
Publishing
Co.,
1975,
pp
45-52
3.
Cohn
DV,
Chu
LLH,
Hamilton
JW,
MacGregor
RR:
The
chemistry,
synthesis
and
intracellular
processing
of
parathyroid
hormonal
peptides.
Proceedings
of
the
Fifth
International
Congress
of
Endocrinology,
Hamburg,
July
18-24,
1976.
Amsterdam,
Excerpta
Medica,
(In
press)
4.
Cohn
DV,
Hamilton
JW:
Newer
aspects
of
parathyroid
chemistry
and
physiology.
Cornell
Vet
66:271-300,
1976
5.
Chu
LLH,
MacGregor
RR,
Cohn
DV:
Energy-dependent
intracellular
trans-
location
of
proparathormone.
J
Cell
Biol
72:1-10,
1977
6.
MacGregor
RR,
Chu
LLH,
Cohn
DV:
Conversion
of
proparathyroid
hormone
to
parathyroid
hormone
by
a
particulate
enzyme
of
the
parathyroid
gland.
J
Biol
Chem
251:6711-6716,
1976
7.
Chu
LLH,
MacGregor
RR,
Liu
PI,
Hamilton
JW,
Cohn
DV:
Biosynthesis
of
proparathyroid
hormone
and
parathyroid
hormone
by
human
parathyroid
glands.
J
Clin
Invest
52:3089-3094,
1973
8.
Mollenhauer
HH:
Plastic
embedding
mixtures
for
use
in
electron
microscopy.
Stain
Technol
39:111-114,
1964.
9.
Reynolds
ES:
The
use
of
lead
citrate
at
high
pH
as
an
electron-opaque
stain
in
electron
microscopy.
J
Cell
Biol
17:208-212,
1963
10.
Watson
ML:
Staining
of
tissue
sections
for
electron
microscopy
with
heavy
metals.
J
Biophys
Biochem
Cyto
4:475-478,
1958
11.
Hamilton
JW,
Spierto
FW,
MacGregor
RR,
Cohn
DV:
Studies
on
the
biosynthesis
in
vitro
of
parathyroid
hormone.
II.
The
effect
of
calcium
and
magnesium
on
synthesis
of
parathyroid
hormone
isolated
from
bovine
parathyroid
tissue
and
in-
cubation
medium.
J
Biol
Chem
246:3224-3233,
1971
12.
Ravazzola
M:
Golgi
complex
alterations
induced
by
X537A
in
chief
cells
of
rat
parathyroid
gland.
Lab
Invest
35:425-429,
1976
13.
Roth
SI:
The
ultrastructure
of
primary
water-clear
cell
hyperplasia
of
the
para-
thyroid
glands.
Am
J
Pathol
61:233-248,
1970
Vol.
87,
No.3
TRIS
AND
HUMAN
PARATHYROID
561
June1977
14.
Habener
JF,
Kemper
B,
Potts
JT
Jr,
Rich
A:
ProparathNToid
hormone:
Biosynthesis
by
human
parathyroid
adenomas.
Science
178:630-633,
1972
15.
Physicians
Desk
Reference,
Twenty-seventh
edition.
Oradell,
N.J.,
NMedical
Econom-
ics
Co.,
1973,
pp
535-536
16.
Friedman
Z,
Hanley
WB,
Radde
IC:
Ionized
calcium
in
exchange
transfusion
with
THANM-buffered
ACD
blood.
CMA
Journal
107:742-745,
1972
17.
Baum
JD,
Robertson
NRC:
Immediate
effects
of
alkaline
infusion
in
infants
with
respiratory
distress
syndrome.
J
Pediatrics
87:255-261,
1974
18.
Nahas
GG:
Hepatic
injury
related
to
tromethamine.
JAMA
206:1793,
1968
19.
Allen
DE,
Baker
DJ,
Gillard
RI:
Metal
complexing
by
Tris
buffer.
Nature
(Lond)
214:906-907,
1967
20.
Hanlon
DP,
Watt
DS,
Westhead
EW:
The
interaction
of
divalent
metal
ions
with
Tris
buffer
in
dilute
solution.
Anal
Biochem
16:225-233,
1966
21.
Goldenberg
VE,
Wiegenstein
L,
Hopkins
GB:
Hepatic
injury
associated
with
tro-
methamine.
JAMA
205:81-84,
1968
2.2.
Eagle
H:
Buffer
combinations
for
mammalian
cell
culture.
Science
174:500-503.
1971
23.
Garrard,
WT:
Selective
release
of
proteins
from
Spirillum
itersonji
by
Tris(hvdroxvmethvl)aminomethane
and
ethvlenediaminetetraacetate.
J
Bacteriol
105:93-100,
1971
24.
Fedvnskyj
NM,
Beck
LV:
Tris
(hvdroxymethyl)
aminomethane
(THAM)
induced
stimulation
of
insulin
release
by
islets
of
Langerhans
previously
isolated
from
rat
pancreas.
Diabetes
19:559-562,
1970
Acowledments
The
authors
gratefully
acknowledge
the
technical
assistance
of
Cecilia
S.
Maben
and
william
J.
Bopp.
562
McGREGOR
ET
AL.
American
Journal
of
Pathology
All
illustrations
are
electron
micrographs
of
sections
stained
with
lead
citrate
and
uranyl
acetate.
.>
t
<-
.-.
XI
Ii
_-
_r
AKF
.%
tfi
ixL
Figur
1-Normal
parathyroid
gland,
3
hours
postmortem,
before
incubation.
Mitochondria
(M),
Golgi
apparatus
(G),
secretory
granules
(S),
and
other
cellular
structures
appear
normal
except
for
focal
mild
degenerative
changes.
L
=
lipid.
(x
9520)
F
2-Normal
parathyroid
gland,
same
as
Figure
1,
following
incubation
for
2
hours
in
control
MIEM
solution.
All
cellular
structures,
including
mitochondria
(Ml),
Golgi
apparatus
(G),
and
secretory
granules
(S),
appear
normal.
(x
14,000)
F
49-
,
t
,.-
..V'
Is
".
1.
v
3
D.~~~~~~~~~~~~~~~~~~~~~~~~J
Figure
3-Normal
parathyroid
gland,
same
as
Figure
1,
following
incubation
for
1
hour
in
control
MEM
solution
then
1
hour
in
the
same
buffer
containing
50
mM
Tris.
Multiple
membrane-limited
vacuoles
(V)
0.15
to 1.7
,
in
diameter
appear
to
be
concentrated
in
the
region
of
the
Golgi
apparatus,
occasionally
contain
membranous
(m)
or
secretory
granule-like
(s)
material,
and
sometimes
coalesce
with
an
adjacent
vacuole.
Normal
secretory
granules
are not
identified.
Other
structures,
including
mitochondria
(M),
appear
normal.
L
=
lipid.
(x
15,000)
Figure
4-Normal
parathyroid
gland,
same
as
Figure
1,
following
incubation
for
1
hour
in
control
MEM
solution,
then
1
hour
in
the
same
buffer
containing
diethylamine. Multiple vesicles
(V)
similar
to
those
resulting
from
incubation
with
Tris
are
suggestive
of
Golgi
origin
and
occasionally
contain
membranous
material
(m).
(x
28,000)
Fgw
5-Chief
cell
hyperplasia
of
parathyroid
gland,
surgically
excised;
follwing
incubation
for
2
hours
in
control
EB
solution.
Cellular
structures
appear
intact.
L
=
lipid.
(x
11,480)
Fe
6-Chief
cell
hyperplasia
of
parathyroid
gland,
same
as
Figure
5,
except
following
incubation
for
1
hour
in
control
EB
solution
then
1
hour
in
the
same
buffer
containing
50
mM
Tris.
Multiple
vesicles
(V)
present
in
the
cytoplasm
are
suggestive
of
Golgi
origin
and
are
similar
to
those
seen
in
Figures
3
and
4.
Other
cellular
structures
appear
intact
(x
14,000)
Figure
7-Oxyphil
adenoma
(functioning)
of
parathyroid
gland,
surgically
excised;
before
incubation.
Oxyphil
cells
(0),
transitional
oxyphil
cells
(T),
and
rare
chief
cells
(C)
are
present.
Mitochondria
(M),
Golgi
apparatus
(G),
secretory
granules
(S),
and
other
cellular
structures
appear
intact.
(x
4620)
Figure
8-Oxyphil
adenoma
(functioning)
of
parathyroid
gland,
same
as
Figure
7,
following
incubation
for
1
hour
in
control
EB
solution,
then
1
hour
in
the
same
buffer
containing
50
mM
Tris.
Multiple
vesicles
(V)
present
in
the
cytoplasm
are
suggestive
of
Golgi
origin
and
are
similar
to
those
seen
in
Figures
3,
4,
and
7.
Secretory
granules
are
not
identified.
Mitochondria
(M)
and
other
cellular
structures
appear
intact.
(x
9520)
42-
Fge
9-Chief
cell
adenoma
of
parathyroid
gland,
surgically
excised;
before
incubation.
Mitochondria
(M),
Golgi
apparatus
(G),
and
other
cellular
structures
appear
intact.
(x
14,000)
Fe
10-Chief
cell
adenoma
of
parathyroid
gland,
same
as
Figure
9,
following
incubation
for
2
hours
in
control
EB
solution.
Mitochondria
(M),
Golgi
apparatus
(G),
secretory
granules
(S),
and
other
cellular
struc-
tures
appear
intact.
(x
9520)
V
R
Figure
11-Chief
cell
adenoma
of
parathyroid
gland,
same
as
Figure
9,
following
incubation
for 1.5
hours
in
control
EB
solution
then
0.5
hour
in
the
same
buffer
containing
50
mM
Tris.
There
is
focal
vacuolar
distension
of
Golgi
apparatus
(G)
in
the
lower
cell
and
more
scattered
small
vesicles
(V)
in
the
upper
cell.
Secretory
granules
are
not
identified.
Mitochondria
(M)
and
other
cellular
structures
appear
intact.
(x
22,400)
Figure
12-Chief
cell
adenoma
of
parathyroid
gland,
same
as
Figure
9,
following
incubation
for
1
hour
in
control
EB
solution,
then
1
hour
in
the
same
buffer
containing
50
mM
Tris.
There
is
vacuolar
distension
of
Golgi
apparatus
(G)
and
scattered
vesicles
(V),
but
these
changes
are
greater
in
degree
and
extent
than
that
in
Figure
12.
Secretory
granules
are
not
identified.
Mitochondria
(M)
and
other
cellular
structures
appear
intact.
(x
15,000)
