Role of Lysosomes in the Pathogenesis of
Splanchnic Ischemia Shock in Cats
By Thomas M. Glenn, Ph.D. and Allan M. Lefer, Ph.D.
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ABSTRACT
Splanchnic arterial occlusion (SAO) for. 2 hours followed by release of the
occlusion in cats produced a lethal shock state characterized by cardiovascular
collapse. Release of the occlusion resulted in a 45% fall in mean arterial blood
pressure within 15 minutes; postrelease survival of these animals was 46
minutes. The plasma of cats with SAO shock exhibited a 3- to 4-fold increase in
activities of the lysosomal enzymes /3-glucuronidase and cathepsin, accompanied by accumulation of a myocardial depressant factor. Plasma from cats
treated with methylprednisolone (20 mg/kg) prior to occlusion did not have
significant levels of myocardial depressant factor nor significant increases in
plasma lysosomal enzyme activity. Furthermore, the fall in mean arterial blood
pressure in steroid-treated animals was significantly smaller when the occlusion
was released and postrelease survival time was significantly longer. Pancreatic
lysosomes from cats with SAO exhibited a marked increase in fragility as
indicated by a reduction in total lysosomal enzyme activity and an increase in
the percent of free enzyme activity, compared to lysosomes from cats with
sham SAO or steroid-treated cats. These data indicate that the biochemical and
hemodynamic alterations present in SAO-induced shock may be related to the
disruption of pancreatic lysosomes and that glucocorticoids can markedly alter
the course of this shock, possibly by decreasing the sensitivity of pancreatic
lysosomes to splanchnic ischemia.
ADDITIONAL KEY WORDS
pancreas
methylprednisolone
mean arterial blood pressure
myocardial depressant factor
cathepsin
/3-glucuronidase
electron microscopy
• Occlusion of the major vessels supplying
the splanchnic bed and release of the occlusion after several hours result in the production of a severe shock state (splanchnic
ischemia shock) both in man (1) and in
experimental animals (2). Shock caused by
splanchnic arterial occlusion (SAO) is characterized by a cardiovascular collapse similar to
that seen in the late stages of hemorrhagic and
septic shock (3). Therefore, previous investi-
From the Department of Physiology, University of
Virginia School of Medicine, Charlottesville, Virginia
22901.
This study was supported by U. S. Public Health
Service Grant HE-09924 from the National Heart
Institute and by a grant from the American Heart
Association. DT. Glenn is a Postdoctoral Trainee of the
National Heart and Lung Institute. Dr. Lefer is an
Established Investigator of the American Heart
Association.
Received July 23, 1970. Accepted for publication
September 10, 1970.
R u t
tfCb,
Vol. XXV11, Nor*mttr 1970
gators have attempted to implicate hypovolemia and endotoxemia as the primary lesions
in the pathogenesis of SAO shock (2, 4, 5).
However, these factors cannot explain the
cardiovascular collapse seen in SAO shock.
Several workers (1, 2, 6) have recently
demonstrated that a significant degree of
myocardial impairment occurs during SAO
shock and that this impairment may contribute significantly to the lethality of the shock
state. It has been previously reported that a
myocardial depressant factor (MDF) accumulates in the plasma of animals subjected to
shock by hemorrhage, endotoxin, or bowel
ischemia (7). Furthermore, it has been shown
that MDF exerts a marked cardiotoxic effect
on the isolated heart ( 8 ) , the isolated
papillary muscle (9), and in the intact animal
(10). Thus the negative inotropic action of
MDF may contribute to the impairment of
cardiac function characteristically seen in a
783
GLENN, LEFER
784
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number of shock states, including SAO shock
(7).
MDF has been shown to be a peptide
having a molecular weight of 800 to 1000
(11). Previous studies have shown that either
the activators or the precursors of MDF arise
from the ischemic splanchnic region, probably
from the pancreas (9), and are transported
via the lymph to the systemic circulation (12).
Although the precise cellular events which
initiate the formation of MDF are not well
defined, hypoperfusion of the splanchnic bed
appears to be the common denominator of all
of the shock states in which MDF has been
found (7). It has also been suggested that
activation of lysosomal hydrolases in the
ischemic splanchnic region and their subsequent release into the systematic circulation
may play a role in the development of
irreversibility in a number of shock states,
including SAO shock (13). Furthermore,
plasma accumulation of MDF is associated
with significant increases in plasma lysosomal
enzyme activities in both endotoxin (14) and
hemorrhagic (15) shock.
Pharmacologic concentrations of glucocorticoids are known to protect in many forms of
shock if given early in the shock state (7, 15).
However, the mechanism of the protective
action of steroids in shock has remained
obscure.
The present investigation was undertaken
to: (1) characterize the properties of the
lysosomes of the pancreas during splanchnic
ischemia shock; (2) to determine if any
relationships exist between the activation of
MDF and alterations in the properties of
pancreatic lysosomes in splanchnic ischemia
shock; and (3) to determine the effects of
methylprednisolone, a glucocorticoid, on heart
rate, arterial and venous blood pressures,
MDF production, status of pancreatic lysosomes, and survival in splanchnic ischemia
shock.
pentobarbital (30 mg/kg iv). The right carotid
artery, left femoral artery, and right femoral vein
were cannulated, and mean arterial blood pressure (MABF), central venous pressure, and heart
rate recorded continuously on a Beckman Type
RB dynograph. All animals received heparin
sodium (1,500 U/kgiv).
SPECIAL SURGICAL PROCEDURES
Splanchnic Arterial Occlusion
This procedure consisted of complete ligation
(clamping) of the celiac, superior mesenteric and
inferior mesenteric arteries. The clamps were
removed 2 hours later, and MABP then declined
rapidly. When the MABP had declined to 60 mm
Hg, 25 to 35 ml of blood was drawn through the
carotid arterial cannula, and the experiment was
terminated. Blood samples to determine plasma
lysosomal enzyme activities were taken at fixed
intervals throughout the experiment. No blood or
fluid replacement was given to replace blood from
sampling. Postrelease survival time is defined as
the rime from removal of the arterial clamps to
the time at which the MABP spontaneously
declined to 60 mm Hg.
In some of the cats, splanchnic arterial
occlusion was carried out as previously described,
but the clamps were not removed at the end of
the 2-hour occlusion period. At this time, 25 to 35
ml of blood was drawn for MDF determination,
and the experiments were terminated. These cats
are termed SAO (no release) cats.
A third group of cats was subjected to the same
surgical procedures except that the arteries were
isolated but not clamped. These animals are
termed sham SAO cats.
Adrenolecromy
Four cats were bilaterally adrenalectomized
under sterile conditions according to previously
described techniques (16), given dexamethasone
(1 mg/day) for the first 2 postoperative days, and
maintained on 0.9$ NaCl and Purina cat chow ad
libitum for 13 days. At this time, the animals
were anesthetized and a rapid pancreatectomy
was performed.
Stsrold-Trasted Animob
ANIMALS
Methylprednisolone1 (20 mg/kg iv) was given
30 minutes prior to splanchnic arterial occlusion
or sham splanchnic arterial occlusion. An additional dose of the steroid (20 mg/kg) was
injected intravenously at the end of the 2-hour
occlusive period just prior to removing the arterial
clamps. This second dose was given to maintain
high blood levels of the steroid during the
postrelease period.
Healthy, parasite-free adult cats of either sex
weighing 2.4 to 3.7 kg were employed in this
study. The animals were anesthetized by sodium
1Solu-Medrol generously supplied by the Upjohn
Co., Kalamazoo, Michigan.
Methods
CirCuUHcn Rciurcb, Vol. XXVll, November 1970
785
LYOSOMES IN SHOCK
HOMOGENIZATION AND FRACTION ATION OF
THE PANCREAS
Preparation of Lysosomal Fraction*
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At the termination of each experiment, the
pancreas was rapidly excised, placed in cold
sucrose solution ( 0 . 2 5 M ) , and weighed at 4°C.
A 1:10 (vv/v) pancreatic homogenate was
prepared using a low speed Waring blender for
15 to 20 seconds. The homogenates were then
centrifuged at 4°C and 1000 X g for 10 minutes.
The sediment, consisting primarily of nuclear
material and cell debris, was discarded. The
supernatant fluid was centrifuged in a Sorvall
centrifuge at 15,900 X g for 20 minutes (318,000 g X min). An aliquot of the supernatant fluid
was taken for the assay of y3-glucuronidase. The
activity obtained from the S fraction is referred to
as the "free" enzyme activity. The pellet (1),
which consisted of lysosomes and other subcellular particles, was washed twice with cold 0 . 2 5 M
sucrose and then gently resuspended in 15 ml of
cold sucrose ( 0 . 2 5 M ) in a Waring blender.
Thermal Activation of Bound Lyioiomal Enzymat
The lysosomal suspension prepared from fraction L was incubated in a Dubnoff water bath
shaker at 37°C for 150 minutes. Aliquots of the
incubated suspension were taken at selected
intervals (0, 30, 60, 90, 120 and 150 minutes)
and assayed for /3-glucuronidase activity. This
procedure resulted in the thermal activation
(release of free enzyme activity) of the bound or
intralysosomal enzyme activity. In addition, the
time course for lysosomal disruption can be used
as an index of the fragility of the population of
lysosomes. The aliquots were then centrifuged at
15,900 X g for 20 minutes. The supernatant fluid
which resulted from this centrifugation was then
assayed for /3-glucuronidase activity. Total enzyme activity of the pancreatic homogen ate
expressed in terms of activity per milligram of
protein was computed by adding the activity
obtained from the S fraction to that obtained from
the L fraction. Preliminary experiments indicated
that the sum of the S and L fractions was not
significantly different from the total enzyme
activity of the original homogenates.
Steroid-Traated Lysoiomts (in vitro)
A lysosomal pellet prepared from normal cat
pancreas was divided into two fractions and the
fractions resuspended in 0 . 2 5 M sucrose. To the
first lysosomal suspension, methylprednisolone
5 X 10-5 mM/liter was added. This dose of
steroid was calculated on the basis of a dose of 20
mg/kg distributed equally in all the tissues of the
body and is comparable to that used by others
(17). The suspension was then incubated with
the steroid for 1 hour at 4°C. A volume of
CirnUUon Rtsiurck, Vol. XXVII, Norombtr 1970
methylprednisolone vehicle equivalent to that
added to the first suspension was incubated with
the second lysosomal suspension. The composition
of the vehicle in mg/ml is as follows: sodium
biphosphate anhydrous 0.8, sodium phosphate
8.7, methylparaben 1.2, and propylparaben, 0.14.
At the end of the incubation period, the
suspensions were subjected to the standardized
thermal activation test and yS-glucuronidase
activity calculated (see above). In this "manner,
half of each lysosomal pellet served as an internal
control for the other.
PROCESSING OF PLASMA FOR MDF ACTIVITY
Blood samples were centrifuged at 2000 X g
for 20 minutes. The plasma was removed and
placed inside dialysis tubing (Nojax tubing,
viscose process, Union Carbide Corp., 30 mm flat
width) at 4°C under a pressure of 200 mm Hg
for 36 to 48 hours. Ten-ml aliquots of the
ultrafiltrates were then lyophilized to dryness.
The samples were then reconstituted to 20% of
their original volume with distilled water and
fractionated on a gel filtration column (Bio-gel P2, 200-400 mesh) as previously described (11).
Elution of the plasma ultrafiltrates from the
column with Krebs-Henseleit solution minus
glucose yielded five fractions read at 230 m^t, one
of which (the fourth fraction, peak D) accounted
for all the MDF activity of plasma'. All the other
fractions contained negligible MDF activity.
Assays for MDF activity were carried out on
isolated cat papillary muscles according to
previously described techniques (18). Samples
containing MDF activity exerted a prominent
negative inotropic effect on the isolated cat
papillary muscle, and the activities were expressed in MDF units. One MDF unit is equal to
1% decrease in the developed tension of the cat
papillary muscle compared with the developed
tension of the muscle in Krebs-Henseleit solution.
CHEMICAL DETERMINATIONS
Samples of plasma and lysosomal fractions (0.1
ml) were assayed for jQ-glucuronidase activity
according to the method of Talalay et al. (19)
using phenolphthalein glucuronide as substrate.
Specific activities were expressed as the number
of micrograms of phenolphthalein liberated per
milligram of protein per hour at 37°C.
The method of Anson (20) was employed to
determine plasma cathepsin activity using bovine
hemoglobin as substrate. Cathepsin specific
activities are expressed as the number of
mi ili equivalents of tyrosine released per milligram
of protein per hour at 37°C. Cathepsin determinations could not be carried out on pancreatic
lysosomal fractions because of interference from
other pancreatic proteases.
786
GLENN, LEFER
TABLE 1
Mean Arterial Blood Pressures and Survival Times of Cats with Splanchnic Artery Occlusion
SAO
Variable
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MABP (mm Hg)
(initial)
MABP (mm Hg)
(at release
or sham release)
MABP (mm Hg)
(15 min
after release)
MABP (mm Hg)
(end of expt)
Postrelease
survival time
(min)
Sham SAO
+flteroid#
SAO
Sham SAO
SAO
(9)
(no release)
W
159 ± 6.3
156 ± 5.4
158 ± 6.6
157 ± 7.0
162 ± 5.7
142 ± 7.4
136 ± 3.8
146 ± 2.1
131 ± 9.7
145 ± 4.0
131 ± 7 . 3
75 ± 4.2
t
128 ± 11.2
119 ± 4.8
(6)
L09 ± 6.1
60+
t
107 ± 7 . 8
§
42.2 ± 6.0
t
§
'%)'
60 *
179 ± 23.7
All values are means ± SE. Number in parentheses is the number of cats in each group.
•The steroid was methylprednisolone. tExperiments terminated just before release of clamps
(120 minutes after occlusion). ^Experiments terminated when MABP declined to 60 mm Hg.
§Sham-operated cats were matched in time to their appropriate experimental groups.
Protein determinations were carried out employing a micro-biuret technique with the
absorbance read at 300 mfi and calibrated with
micro-Kjeldahl. determinations.
ELECTRON
MICROSCOPY
Small sections of pancreatic tissue (2 to 3
mm2) were fixed in 4% cacodylate-buffered glutaraldehyde (pH 7.4). Forty-micron thick sections
were then made from the larger sections on a
freezing microtome and collected in cold cacodylate buffer. The sections were then incubated
at 37°C in a Gomori medium (pH 6.5) for the
presence of acid phosphatase (a lysosomal
marker), rinsed in cold sodium acetate buffer
(pH 6.5 ), and postfixed in cacodylate-buffered
1% osmium tetraoxide for 1 hour according to the
method of Miller and Palade (21). The sections
were then dehydrated in ethyl alcohol and
embedded in Epon. Ultra-thin section (600A)
were prepared with uranyl acetate and lead
citrate to enhance their contrast of the embedded sections. Lysosomal fractions (L) were
treated in essentially the same manner as the
pancreatic sections.
Results
A summary of the changes in systemic mean
arterial pressure and postrelease survival times
for all five groups of cats studied are shown in
Table 1. Mean arterial blood pressure decreased slightly over the 2-hour occlusion
period in all of the groups studied. However,
release of the occlusion in SAO cats resulted in
a significant (P<0.001) fall of 45% in MABP;
whereas SAO cats treated with methylprednisolone exhibited only a 17$ decrease in MABP.
Cats with sham SAO with and without steroid
treatment had no significant changes in MABP
during the same interval. The modification of
the initial hemodynamic response to release in
the shocked cat by methylprednisolone does
not seem to be dependent on the ability of the
steroid to exert any specific changes in
hemodynamic function since the steroid produced no significant changes in MABP, central
venous pressure, or heart rate.
In addition, previous steroid treatment of
SAO cats resulted in a fourfold increase in
postrelease survival ( P < 0.001). T h u s , t h e
pharmacologic doses of methylprednisolone
employed in SAO cats significantly modified
the initial hemodynamic response to the
release of the occlusion and also increased the
postrelease survival time of these animals.
The plasma MDF activities for all five
groups of cats are summarized in Figure 1.
Previous studies have mdkated tbst an inverse
relationship exists between plasma MDF
concentration and survival (7). Tin's relationGircmloion RUNRCJJ, Vol. XXVII, N n b f 1970
787
LYSOSOMES IN SHOCK
-
60 50 40 30 50 -
-I-
10 -
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r
8
Sham
SAO
iI
I
1n ft
M ui
n•
u ui
+
SAO
SAO
Sham
SAO
SAO
4
4rf n
FIGURE 1
Bar graph of plasma myocardial depressant activity
in column ekiates expressed in MDF (myocardial depressant factor) units, obtained from all five groups
of cats studied. Height of the bars indicates mean
MDF activity, and SE is indicated. Number inside
bar is number of samples measured. One MDF unit
is equal to a 1% decrease in the developed tension of
an isolated cat papillary muscle under standardized
conditions (see Methods).
ship was also found in the present investigation. Whereas MDF activity in plasma from
SAO cats was very high, that in plasma from
steroid-treated cats subjected to the same
degree of SAO was not significantly different
from that found in sham SAO and steroidtreated sham SAO cats. In addition, plasma
from cats subjected to SAO without release of
the occlusion did not have significant MDF
activity. Thus, MDF appears to be formed in
the ischemic splanchnic region during the
occlusive period and probably enters the
circulation in large quantities only after
release of the occlusion.
. Two typical column elution patterns of
plasma ultrafiltrates are illustrated in Figure 2.
The upper panel shows that peak D eluted
from an SAO cat contained essentially all the
MDF activity (63 MDF units) present in the
plasma. The peak D of an ultrafiltrate from a
steroid-treated cat subjected to SAO was
smaller in area and contained very low MDF
activity (17 MDF units) compared with the
SAO sample. No appreciable MDF activity
CircmUaion Riitorcb, Vol. XXVII, Novrmifr 1970
was found in any of the other four peaks
eluted from the ultrafiltrates in either sample.
Figures 3 and 4 summarize the alterations in
plasma /3-glucuronidase and cathepsin activities during SAO shock; both lysosomal enzymes showed similar plasma activity patterns. There was a significant increase in the
activities of both enzymes within 15 minutes
after arterial occlusion. This high level of
activity was fairly well maintained over the 2hour occlusive period. An additional rise in
the plasma activities of these enzymes occurred following release of the occlusion. This
second rise presumably indicates the washout
of enzyme that has accumulated in the
Splanchnic Artery Occlusion
A
B
C
D
8 - 2 6
63
Spbnchnic Artery Occlusion+Methytpr««solone ( * 4 6 0 )
A
B
C
D
E
10
20
17
TUBE NUMBER
Column elution patterns of typical plasma ultrafiltrates obtained from a cat subjected to splanchnic
artery occlusion and a methylprednisolone-treoted cat
subjected to splanchnic arterial occlusion. Peak D at
top contained all the MDF activity found in the
plasma; that at bottom is much smaller and did not
contain significant MDF activity. Note that methylprednisolone prevented the appearance of MDF in
plasma following splanchnic arterial occlusion of 2hour duration.
788
GLENN, LEFER
5
it
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Q. g
2
0
•
• Splanchnic orhiry occlusion (N • 7)
o
o Sham Splanchnic artery occkulon (N« 6 )
W
60
90
120
150
Time (minutes)
FIGURE 3
Graph of plasma fi-glucuronidase activity in cats subjected to splanchnic arterial occlusion
and sham splanchnic arterial occlusion. Occlusion of celiac, superior, and inferior mesenteric
arteries at first arrow and release of the occlusion at the second arrow. Means and SE are
shown.
Time (minutes)
Graph of plasma cathepsm activity in cats subjected to splanchnic arterial occlusion and
sham splanchnic arterial occlusion. Occlusion of celiac, superior, and inferior mesenteric arteries is noted by the first arrow and release of the occlusion by the second arrow. Means and
SE are shown.
ischemic splanchnic bed during the period of
occlusion. The rise in /3-glucuronidase activity
peaked within 30 minutes after release but
the cathepsin activity continued to increase
R , i ,
anb, Vol. XXVII, November 1970
789
LYSOSOMES IN SHOCK
TABLE 2
Plasma Beta Gkwuronidase Activity
Initial
(O min)
Prertleaie
(120 min)
Postrtleaie
(US min)
Tennlnal
37.7±4.3
55.0 ±10.6
66.7 ±14.4
33.4 ± 4.3
36.7 ± 5.4
e
30.5 ± 3.4
25.0 ±9.2
29.6 ± 5.3
75.5 ± 11.4
82.9 ± 9.8
5
27.6 ± 2.8
30.2 ±4.3
27.0 ±4.1
34.6 ± 2.9
5
29.5 ± 3.1
30.0 ±8.7
29.6 ±4.1
30.6 ± 4 . 6
Group
N
Sham SAO
SAO (no release)
SAO
SAO +
methylprednisolone
Sham SAO +
methylprednisolone
5
4
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p-glucuronidase activity is expressed as micrograms of phenolphthalein XlO~2 released per
milligram protein per hour at 37°C. All values are means ± SE, N = the number of animals
in each group.
TABLE 3
Plasma Cathepsin Activity
Initial
(Omin)
Prerrelease
(120 min)
Pastrelease
(135 ml n j
Terminal
6
4
6
7.1 ± 1.4
5.5 ±1.8
5.9 ± 0.8
7.2 ±1.2
10.9 ±0.9
9.5 ±1.2
6.4 ± 1.0
8.7 ± 2.8
11.8 ± 2.1
21.1 ±3.7
5
4.8 ±1.1
6.0 ±1.9
4.8 ± 1.5
3.6 ± 0 . 3
5
5.0 ± 0.8
6.6 ± 0.8
6.3 ± 1.1
3.3 ± 0.9
Group
u
Sham SAO
SAO (no release)
SAO
SAO +
methylprednisolone
Sham SAO +
methylprednisolone
Cathepsin activity is expressed as milliequivalents of tyrosine X 10-* released per miHigrain
protein per hour at 37° C. All values are means ± SE. N— number of animals in each group.
throughout the entire postrelease period. Final
enzyme activities were 3 to 4 times the preSAO values.
Plasma activities for /3-glucuronidase and
cathepsin for all five groups of cats are
summarized in Tables 2 and 3. Two hours
after clamping, there was a significant rise in
the activities of both lysosomal enzymes in
plasma from SAO cats with and without
release of the vascular clamps. The plasma
activities of both enzymes were not significantly altered in the other three groups of animals
at any of the times at which they were
measured. Thus, methylprednisolone treatment prevented the increases in lysosomal
enzyme activities in the plasma during the
entire course of SAO shock. The significant
increases in lysosomal enzyme activity observed in SAO shock probably reflects the
degree of lysosomal disruption in the ischemic
splanchnic bed. Although splanchnic ischemia
Circulnitm Ruttrcb,
Vol. XXVII, Norrmltr
1970
was produced by clamping of the splanchnic
arteries, the steroid appeared to interrupt the
normal response to ischemia, namely, the
disruption of splanchnic lysosomes with the
subsequent release of their enzyme content
into the systemic circulation.
To relate possible changes in pancreatic
lysosomes with the biochemical and hemodynamic changes in SAO shock, lysosomal
fractions of pancreas were prepared and
studied. Figure 5 represents electron micrographs which show the presence of lysosomes
in the fractions isolated from the normal cat
pancreas. A, an electron micrograph of a
portion of a pancreatic acinar cell, depicts
lysosomes containing the stained acid phosphatase particles, interspersed with zymogen
granules, and distributed in a perinuclear
fashion. An electron micrograph of a "lysosomal pellet" prepared from the supernatant
fraction of a pancreatic homogenate is shown
GLENN, LEFER
790
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if* .
r JW
I? ;
- v
f
;
:,
-••
* •
1 < 'I • <
'
5
FIGURE S
Electron micrographs of a portion of an adnar pancreatic cell (A), supernatant fraction of
tissue homogenate (B and C) and same fraction incubated for 120 minutes at 37°C (D).
Magnifications: A, B and T>, 25,620X; C, 76,880X- All sections were fixed in glutaraldehyde
and incubated for acid phosphatase. Acid-phosphatase-positioe granules can be seen scattered in some of the ribosomes of the rough endoplasmic reticuhun and concentrated in the
lysosomes. The lysosomes of the pancreas are tmrilar in appearance to lysosomes in other
tissues. The acid-phosphatase-positive granules can be seen in the homogenate fraction within
intact lysosomes. After the lysosomes were disrupted by thermal agitation (D), no acid phosphatase can be seen within "ghost" membranes.
in B. The pellet contains several lysosomes,
zymogen granules, a smaller number of
mitochondria, and other subcellular particles.
C is an electron micrograph of the lysosomal
pellet shown in B photographed at higher
magnification. The five lysosomes in this
micrograph are characterized by the intensity
and distribution of the acid-phosphatase
positive particles within the lysosomes and the
vacuoles which are prominent in several of the
lysosomes pictured. To disrupt the lysosomes
and release their internal enzymes, the lysosomal pellet was subjected to a thermal
activation test. D is an electron micrograph of
the same lysosomal pellet after it was
subjected to the disruptive effect of 120
OraUtion Resotrcb, Vol. XXVU, Nortmhtr 1970
LYSOSOMES IN SHOCK
791
Shorn SAO
Sham SAO
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Graph showing typical disruption curves for lysosomal
pellets. The lysosomal suspensions were incubated at
37°C for 150 minutes to release bound enzymes
from intact lysosomes. Thermal activation of bound
enzymes occurred in 60 minutes within the pellet
from a sham SAO cat compared with 120 minutes in
the pellet from a methylprednisolone-treated sham
SAO cat. Only 65% of the bound lysosomal enzyme
was released in the steroid treated cat at 60 minutes
of incubation at a time when lysosomes of sham SAO
cat were maximally released.
minutes of agitation at 37°C. This micrograph
depicts many intact membranes which do not
appear to contain any significant quantities of
intragranular material and probably represent
"ghost" membranes from lysosomes, mitochondria and other subcellular particles.
' Thermal activation of lysosomes results in
the release of intralysosomal enzymes, the rate
of which serves as an index of their fragility.
Figure 6 shows the rate of release of /?glucuronidase from a lysosomal pellet. Maximal release of /3-glucuronidase activity occurred much more rapidly in the lysosomal
pellet obtained from a sham SAO cat than
that from a steroid-treated sham SAO animal.
After 60 minutes of incubation, maximal
enzyme release had taken place in the pellet
from the untreated cat. At this time, however,
only 65% of the bound /3-glucuronidase activity
had been liberated from the pellet obtained
from the methylprednisolone-treated cat. ExCircuUaion Rtsurcb, Vol. XXVII, NotHnbir 1970
pressed differently, only 10 minutes of incubation were required to release 5O!6 of the (iglucuronidase activity from the sham SAO
lysosomal pellet, whereas 30 minutes were
required to release 50S> of the enzyme activity
in the pellet from the steroid-treated sham
SAO cat. Thus administration of methylprednisolone in vivo appears to markedly increase
the stability of pancreatic lysosomes.
The time required for peak release of
/3-glucuronidase activity from the lysosomal
pellets in the five groups of animals studied
are summarized in Figure 7. There are no
significant differences in the time to peak
enzyme release from the lysosomal pellets
obtained from the three groups of animals not
given steroid (i.e., sham SAO, and SAO with
and without release). However, methylprednisolone treatment in either sham SAO or SAO
cats markedly increased the time required for
PANCREATIC LYSOSOMAL FRAGILITY
Bar graph illustrating the time required for 100% disruption of pancreatic lysosomal fraction obtained from
all floe groups of cats studied. Height of bars indicates the mean time required for maximal activation
of lysosomal enzyme by thermal activation, and SE
is shown; number in bar is the number of preparations studied. The times required for maximal activation of lysosomal fraction from cats with sham
SAO, SAO and SAO (no release) are not significantly
different. Methylprednlsolone treatment of cats with
shock and those without shock produced a significant
(P < 0,001) increase in the time required for maximal lysosomal disruption.
GLENN, LEFER
792
TABLE 4
Influence of Splanchnic Arterial Occlusion, Methylprednisolone, and Adrenalectomy on Pancreatic Lysosomal Enzyme Activity
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Group
N
Total
fi-glucuronid&M
activity
Sham SAO
SAO (no release)
SAO
SAO +
methylprednisolone
Sham SAO +
methylprednisolone
Adrenalectomy
5
4
5
100.9 ± 7.0
39.7 ± 7.9
53.7 ± 5.7
13.9±2.9
27.0 ± 3.2
27.4 ± 3.5
86.1 ± 3.8
73.0 ± 2.4
72.6 ± 3.7
5
60.9 ± 9.5
13.8 ± 3.6
86.2 ± 3.5
5
4
52.2 ± 6.6
103.6 ±12.8
16.9 ± 2.2
23.3 ± 2.2
83.1 ±2.2
76.7 ± 2.5
P«rcetat of total activity
Free
Bound
/3-glucuronidase activity is expressed as micrograms of phenolphthalein XlO~2 released per
milligram protein per hour at 37°C. N = number of animals in each group. All values are
means ± SE.
their respective lysosomes to be thermally
activated. These data indicate that methylprednisolone administration resulted in an
increased stabilization of pancreatic lysosomes. This finding may explain the decreased
sensitivity of the steroid-treated cats to the
trauma of splanchnic ischemia.
Total pancreatic /3-glucuronidase content as
well as extralysosomal (free) and intralysosomal (bound) fractions were calculated. The
values obtained are shown in Table 4. A
significant increase in free /3-glucuronidase
activity occurred in animals subjected to SAO
with and without release. In addition, there
was a marked decrease in the total pancreatic
/3-glucuronidase activity in these two groups
of SAO cats compared to the total activity
observed in sham SAO cats. The decreased
total pancreatic enzyme activity present in the
SAO cats can probably be accounted for by
the marked increase in plasma lysosomal
enzyme activity seen in these cats. Thus, a
large number of these lysosomes had already
released much of their enzymes during shock
and could therefore not release as much as
lysosomes from nontraumatized cats.
Methylprednisolone pretreatment of the
SAO animals prevented the increase in free
enzyme activity. In fact, free lysosomal
enzyme activity in both groups of steroidtreated animals was not significantly different
from that seen in sham SAO cats. The total
pancreatic lysosomal enzyme activity of both
groups of steroid-treated cats was significantly
lower than that seen in cats not treated with
steroids, regardless of whether they were
subjected to shock. This suggests that methylprednisolone increased the stability of the
pancreatic lysosomes, and maximal release of
enzyme activity could not occur in these
lysosomes.
Since methylprednisolone altered some of
the basic characteristics of pancreatic lysosomes, pancreatic lysosomes obtained from
chronically adrenalectomized cats were also
studied. The data indicate that pancreatic
lysosomes obtained from adrenalectomized
cats are less stable than normal. This is
indicated by the fact that the percent of free
/}-glucuronidase was 67% higher than in
normal cats even though the total /9-glucuronidase activity was unchanged. Quanitatively,
the free pancreatic /3-glucuronidase activity in
adrenalectomized cats was comparable to that
found in the pancreatic lysosomes of cats after
splanchnic arterial occlusion. These findings
indicate that large doses of exogenous glucocorticoid can markedly alter the response of
pancreatic lysosomes to splaachBic ischemia
shock, whereas chronic deprivation of glucocorticoids can result in an increased fragility
of paacreatic lysosames.
Moreover, incubation of isolated pancreatic
OraAaion Rttartb, Vol. XXVU. Nortnbf
1970
LYSOSOMES IN SHOCK
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lysosomes from control cats with concentrations of methylprednisolone equivalent to
those seen in steroid-treated cats ( 5 x 1 ( H M )
resulted in a degree of lysosomal stabilization
comparable to that seen in the intact animal.
Thermal activation of the in vitro steroidtreated lysosomes yielded /J-glucuronidase
activity (31.0 ±3.5; mean±SE) which was
only a third of that seen in lysosomes treated
with the steroid vehicle (91.1 ±17.9). The
total enzyme activities obtained from vehicleand steroid-treated lysosomes in vitro were
equivalent to those found in the lysosomes of
sham SAO and steroid-treated sham SAO
animals. These data indicate that methylprednisolone can stabilize pancreatic lysosomes in
vitro.
Discussion
The data obtained from this study suggest
that activation of lysosomal hydrolases in the
pancreas is related to the production of a
cardiotoxic factor which contributes to the
cardiovascular collapse observed following
occlusion of the arterial vessels supplying the
splanchnic bed.
Previous studies of the pathogenesis of SAO
shock have implicated a number of factors
that may contribute to the lethality of this
shock state. Kobold and Thai (22) have
described the appearance of a vasoconstrictor
peptide in the plasma of animals following
occlusion of the superior mesenteric artery
and suggested that its formation may be the
result of the proteolytic action of pancreatic enzymes. Other investigators have suggested that
the release of bacterial endotoxins from the
ischemic intestine is involved in the pathogenesis of SAO shock (2, 3, 5, 23). Still others
have attributed the lethality of SAO shock to
such diverse factors as hypovolemia (4),
release of vasoactive compounds such as
serotonin, substance P, adenine nucleotides
(24), and proteolytic enzymes (25). However, none of the above factors has been
satisfactorily implicated in the fatal cardiovascular failure of SAO shock.
Recently, several investigators (1, 2, 26)
have observed that a pronounced myocardial
CirctUmo R,iu<ch, Vol. XXVll, Nowtmbtr 1970
793
impairment (as evidenced by a significant
decrease in cardiac output without alterations
in cardiac filling pressures) develops soon
after ooclusion of the superior mesenteric
artery and that the intensity of the cardiac
impairment progresses with continued occlusion. Furthermore, release of the occlusion
itself seems to hasten the cardiovascular
collapse.
Thus Williams et al. (26) demonstrated a
negative inotropic factor in the plasma of dogs
subjected to superior mesenteric artery occlusion which they suggested as the primary
cause of the observed decrease in cardiac
output. They implicated the ischemic gut as
the site of release or production of the factor.
The factor isolated by Williams produces a
negative inotropic response similar to that
obtained with MDF, and may be MDF.
The production of MDF also occurs in a
number of shock states, including those due to
hemorrhage, endotoxin, and pancreatitis in
which the common denominator is hypoperfusion of the splanchnic bed (7). Lefer and
Martin (9) have suggested that MDF may
arise from the ischemic pancreas.
Although splanchnic hypoperfusion appears
to be the initial stimulus for MDF production,
the actual cellular changes leading to the
production of MDF, its activators, or its
precursors have not been clearly defined.
Blockade of the celiac ganglia by local
anesthetics (3) has been shown to reduce the
severity of the shock produced by superior
mesenteric artery occlusion. Wangensifeen et
al. (14) have also demonstrated that celiac
ganglionic blockade results in the maintenance of normal levels of splanchnic blood
flow and prevents the increase in plasma
lysosomal enzymes as well as the plasma
accumulation of MDF in endotoxin shock.
Recent studies have shown that increased
plasma lysosomal enzyme activity in shocked
animals correlates well with the accumulation
of MDF in the peripheral blood (12, 14, 15).
Although many investigators (12-14, 23, 25)
claim that lysosomes play an important role in
the cellular changes leading to the irreversibility of shock states, no convincing demonstra-
794
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tion of a direct toxic effect of lysosomal
enzymes has thus far been reported. Thus
lysosomes of the splanchnic region are known
to be more sensitive to ischemia than the
lysosomes of other regions of the body (27);
and splanchnic ischemia has been shown to
disrupt splanchnic lysosomes (23). Tissue
ischemia and the resulting hypoxia and
acidosis may be responsible for the activation
and release of lysosomal enzymes (13). This
hypothesis was advanced by deDuve (28),
who found that acid conditions accelerated
the release of hepatic lysosomal enzymes. The
activity of the released hydrolases was found
to be optimal at pH 5 (29), a condition
approximating that found in ischemic tissue
(30).
The data presented in this study show that
significant changes in lysosomal enzyme activity and lysosomal integrity take place in
pancreatic lysosomes during the course of
SAO shock. These changes appear to be
temporally related to the subsequent biochemical and hemodynamic alterations occurring
after splanchnic arterial occlusion. The rapid
increase in plasma hydrolase activity indicates
the high degree of sensitivity exhibited by the
splanchnic lysosomes to ischemia. Witte et al.
(30) showed that splanchnic lymph Po2 falls
to less than 8 mm Hg within 10 to 15 minutes
of occlusion of the splanchnic vessels. The fact
that the total pancreatic lysosomal enzyme
activity of the SAO cats was 54$ of that of
sham SAO cats probably reflects the increased
plasma enzyme activity in the SAO cats. The
increase in lysosomal protease activity in the
plasma before the plasma appearance of MDF
supports this contention. Although plasma
lysosomal enzyme activities were significantly
elevated for most of the occlusive period, no
significant MDF activity was found in the
plasma of SAO cats in which the occlusion
was not released.
There are several possible explanations for
these findings. The lysosomal enzymes, and
presumably preformed MDF, are transported
to Ae systemic circulation either via splanchnic collaterals or via the lymphatic system.
Since it would be difficult to transport the
GLENN, LEFER
large molecular weight enzymes' across capillary membranes, they would more likely be
transported by lymphatic vessels. We have
previously demonstrated significant lymphatic
transport of lysosomal enzymes in hemorrhagic shock (12). Furthermore, Nelson et al. (31)
showed a transient spikelike increase in lymph
flow immediately upon initiation of hemorrhage. Lymph flow then gradually declined
(12, 31) upon a sustained splanchnic ischemia
resulting from prolonged oligemia. In the
present study, the rapid increase in plasma
lysosomal enzyme activity may have been due
to this initial increase in lymph flow. In this
regard, intravenous injection of pancreatic
lysosomal enzymes simulating activities observed in SAO shock was sustained over a 2hour period in two normal cats. This corresponds to the period of complete occlusion in
the SAO cats.
In these cats, a large increase in /3glucuronidase activity was observed within 1
minute of intravenous injection of an aliquot
of a pancreatic lysosomal pellet. This increased level of enzyme activity was maintained for 2 hours and decreased only 183S
from the peak value in that time. No
significant changes in MABP, central venous
pressure, or heart rate occurred in these cats.
These data indicate that the /3-glucuronidase
activity can be maintained in the plasma over
periods such as those occurring in the SAO
experiments. These findings support the hypothesis that an initial surge of lysosomal
enzyme activity into the plasma could be
maintained for the period of splanchnic
arterial occlusion.
The transport of lysosomal enzymes and
MDF via the lymphatic system leaves the
question of the lack of high plasma MDF
activities unanswered. There are several possibilities for the low plasma MDF activities in
SAO cats prior to release of the occluded
vessels. One possibility is that there is a
substantial latent period between the release
of lysosomal enzymes and the formation of
MDF. In this regard, a latent period of 2 to 3
hours between splanchnic ischemia and MDF
production has been demonstrated in shock
Riard,
Vol. XXVII, Nonmttr 1970
LYSOSOMES IN SHOCK
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due to endotoxdn (14) or hemorrhage (10).
Another possibility for the failure to accumulate MDF in the plasma may be that the pH
of normal systemic blood (i.e., 7.3 to 7.4)
is too far removed from the optimal pH of
these enzymes (i.e., pH of 5.0 to 5.5).
A third possible reason for the lack of
MDF in systemic plasma may be that MDF,
even if it is formed, may be rapidly inactivated or removed by the normally functioning
kidneys and reticuloendothelial system. These
considerations support the hypothesis that
MDF is produced within the splanchnic
region during splanchnic ischemia shock and
that conditions are not optimal for its
production or accumulation in peripheral
plasma during SAO shock.
Further support for the hypothesis that
MDF is produced within the splanchnic
region can be derived from the work of
Williams et al. (26). These workers found
that a myocardial depressant factor appeared
in the plasma of dogs during the occlusive
period of splanchnic ischemia shock. In their
model, only the superior mesenteric artery was
occluded, whereas in the present study, the
eeliac and superior and inferior mesenteric
arteries were occluded. The possibility exists
that when only the superior mesenteric artery
is occluded, sufficient collateral circulation
developed to ensure the transport of MDF
from the splanchnic bed into the systemic
circulation. Williams et al. (32) have demonstrated that collateral pathways do exist
during experimental superior mesenteric occlusion, The primary collaterals visualized by
arteriography were the eeliac axis and inferior
mesenteric artery. In the SAO shock model
employed in this study, both of these potential
collateral pathways were occluded.
The large decrease in MABP after release of
the occlusion may have resulted from the
washout of large quantities of MDF or some
other agent into the systemic circulation.
Another possibility is that the splanchnic
vasculature, already dilated by vasoactive
agents, becomes engorged with blood after
release of the occlusion. These agents may
Citcidttim R,u~cl>, Vol. XXVII, November 1970
795
arise from the ischemic pancreas, liver, or
intestine.
Data are available to indicate that MDF
can rapidly and severely impair myocardial
contractility. Thus addition of a bolus of MDF
directly into the coronary perfusion medium
of an isolated perfused cat heart decreases
cardiac contractility by 83* within 1 to 2
minutes (8).
Some workers have attributed the protective effect of glucocorticoids in shock to an
alleged direct positive inotropic effect on the
heart (33). Lefer (34) found that cortisol did
not increase the contractile force of either the
isolated cat papillary muscle or the isolated
perfused cat heart. Furthermore, Glenn and
Lefer (15) were unable to demonstrate a
significant increase in contractile force of the
intact cat heart employing doses of methylprednisolone (20 mg/kg) equivalent to those
used in the present study. Lillehei et al. (35)
postulated that, in shock, glucocorticoids
function as alpha-receptor blocking agents
(i.e., vasodilators) but did not provide
evidence for this mechanism. Others have
suggested that the glucocorticoids are beneficial in shock because they potentiate the
vasoconstrictor effect of the catecholamines
(36). However, Kadowitz and Yard (37)
recently showed that doses of cortisol which
significantly protect animals subjected to
endotoxin shock did not enhance the cardiac
response to catecholamines, produce vasodilatation, or act as an alpha-receptor blocking
agent. These investigators proposed that
cortisol might protect by altering the response
of lysosomal enzymes to endotoxin administration.
Weissman and Thomas (17) have demonstrated the abilities of glucocorticoids both in
vivo and in vitro to stabilize lysosomes in
endotoxin and splanchnic ischemia shock.
Glenn and Lefer (15) have proposed that the
protective effect of methylprednisolone in
bemorrhagic shock is based on the ability of
the steroid to stabilize splanchnic lysosomes,
since methylprednisolone-treated cats subjected to hemorrhagic shock had normal plasma
lysosomal enzyme and MDF activities and
GLENN, LEFER
796
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survived longer than vehicle-treated animals
with shock.
The total enzyme activity of the in vitro
methylprednisolone-treated lysosomal fractions was similar to that observed in lysosomes
obtained from intact cats treated with steroid;
whereas the portion of control pancreas
incubated with an equivalent volume of
methylprednisolone vehicle yielded fragilities
and total activities comparable to those found
in lysosomes obtained from cats with sham
SAO. These findings are in agreement with
those of other investigators (13, 17), who
demonstrated the ability of glucocorticoids to
reduce the fragility and free enzyme activity
of lysosomes isolated from animals subjected
to shock.
replacement and survival. Ann Surg 150: 543,
1959.
5. WITZNITZER, T., AND ROZDSJ, R.: Bacterial factor
in shock following superior mesenteric artery
occlusion. Israel Med J 22: 4, 1963.
Depletion of endogenous glucocorticoids by
chronic adrenalectomy resulted in an increase
in lysosome fragility and a higher percent of
free activity than in lysosomes from normal cat
pancreas. The increased fragility of these
lysosomes may provide a partial answer to the
increased sensitivity of adrenalectomized animals to various forms of shock (13). Similar
increases in free lysosomal enzymes in the
tissues of adrenalectomized animals have been
previously reported (38).
10. WANGENSTEEN, S. L., DEHOLL, J. D., KIECHEL,
S. F., MARTIN, J., AND LEFER, A. M.: Influence
Acknowledgment
The authors wish to acknowledge the competent
technical assistance of Mr. Julian B. Martin, Mr.
Thomas F. Inge, Jr., and Mrs. Nancy I. Kelly during
the course of this project.
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Role of Lysosomes in the Pathogenesis of Splanchnic Ischemia Shock in Cats
THOMAS M. GLENN and ALLAN M. LEFER
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Circ Res. 1970;27:783-797
doi: 10.1161/01.RES.27.5.783
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