AMERICAN
Vol.
JOURNAL
228, No.
OF
2, February
PHYSIOLOGY
1975.
Printed
in U.S.A.
Endotoxin-induced
prostaglandin
E and F
release in dogs FRED L. ANDERSON, WILLIAM JUBIZ, THEOFILOS AND HIROSHI KUIDA Cardiovascdar and Metabolism Divisions, Veterans Administration College of bfedicine, Salt Lake City, Utah 84112
shock;
mesenteric
organs;
portal
vein;
renal
acid; indomethacin
SEVERAL VASOACTIVE SUBSTANCES have been implicated in the mechanism of canine endotoxin shock. These include histamine, serotonin, catecholamines, and bradykinin (11, 18, 32, 33). Prostaglandins E and F (PGE and PGF) are vasodepressor and vasopressor lipids, respectively, that are present in many canine tissues. Prostaglandin-like material has been identified in the plasma of dogs with endotoxin shock and acute systemic hypotension but specific identification of these substances has not been made (2, 17). The purpose of this study was to determine PGE and PGF levels by radioimmunoassay in sequential blood samples from the renal and portal veins and aorta during endotoxin shock in dogs. The influence of acetylsalicylic acid and indomethacin pretreatment on endotoxin-induced PGE and PGF reIease was also studied. An analysis of these experiments forms the basis for this report.
l
METHODS
Adult thetized
Hospital, and University of Utah
to avoid anesthesia-induced hypoxemia the animals were ventilated at a constant pressure and rate throughout the experiment with a positive-pressure respirator (Medtronics, Minneapolis) with a gas mixture of either room air (group I) or 20 % 02, 3 % CO2 , and 77 % nitrogen (groups 2, 3, and 4). Two catheters were introduced into the external jugular vein and advanced under fluoroscopy to either the left or right renal vein (RV) and pulmonary, artery (PA), respectively. In 20 of 25 dogs the left renal vein was catheterized. In these dogs the catheter tip was advanced deep into the renal pelvis to avoid contamination with spermatic vein or inferior vena cava blood. A third catheter was introduced into the carotid artery and advanced to the ascending aorta (Ao) for the purpose of sampling blood in as close proximity to the lungs as possible. The abdomen was then entered via a midline incision A small polyethylene cannula was inserted into a branch of the splenic vein and advanced to the portal vein (PV). Correct position of the RV catheter tip in the renal pelvis was confirmed by palpation. A second cannula was inserted into the femoral artery (FA) for the purpose of measuring arterial pressure, All catheters and cannulas were connected to Statham P23Db pressure transducers. Heparin, 10,000 U, was given intravenously to prevent clot formation in the catheters and can&as. Cardiac output (CO) was determined by the dye-dilution method. Indocyanine green dye (5-l 0 mg) was injected into the PA and sampled from the FA through a densitometer (Gilson Medical Electronics, Madison, Wis.) using a constant-rate withdrawal pump (Harvard Apparatus Co., Dover, Mass.). Pressures and the dye curves were recorded on an oscillographic recorder (Minneapolis Honeywell, Denver) Body temperature was not measured or controlled in any of the experimen ts. Blood samples for prostaglandin analysis were handled in the following manner. Each blood sample was immediately transferred to sterile glass tubes containing 0.3 ml of 20 % sodium citrate. After centrifugation for 15 min the plasma was transferred to 5-ml glass containers and frozen immediately. Each sample was analyzed for PGE and PGF by the radioimmunoassay method of Jubiz et al. (16). The antisera used in the present studies were the same that we utilized in that study. Four groups of animals were studied. Group I consisted of five control dogs in which no endotoxin was given. Pressures were recorded continuously for 90 min. Blood samples for prostaglandin levels and CO measurements were ob-
ANDERSON, FRED L., WILLIAM JUBIZ, THEOFKOS J. TSACARIS, HIROSHI KUIDA. Endotoxin-induced ~rostuglandin E and F release in dogs. Am. J. Physiol. 228(Z): 410-414. 1975.-Prostaglandin E and F (PGE and PGF) 1evels in sequential blood samples obtained simultaneously from the renal and portal veins and aorta during endotoxin shock in dogs were determined by radioimmunoassay. Four groups of dogs were studied. In five control dogs in which no endotoxin was given, PGE and PGF levels did not change significantly at 0, 15, 30, 60, and 90 min. In eight dogs given endotoxin alone, PGE and PGF levels did not change in the aorta. In samples taken from the portal vein there was a significant rise in PGE and PGF 15 min after endotoxin, whereas renal vein PGE and PGF did not become significantly elevated until 60 and 90 min after endotoxin. In six dogs pretreated with acetylsalicylic acid and six dogs pretreated with indomethacin, PGE and PGF levels did not change after endotoxin. Indomethacin modified the delayed hemodynamic effects of endotoxin whereas acetylsalicylic acid did not. Neither drug blocked the immediate hemodynamic effects of endotoxin. Endotoxin-induced PGE and PGF release is probably due to increased synthesis. The mechanism whereby synthesis is stimulated and the extent to which vasomotor tone is influenced by PGE and PGF during endotoxin shock cannot be determined from our data. AND
radioimmunoassay; vein; acetylsalicylic
J. TSAGARIS,
mongrel dogs (average weight 23 kg) were aneswith sodium pentobarbita1 (30 mg/kg iv). In order 410
Downloaded from www.physiology.org/journal/ajplegacy at Washington Univ (128.252.067.066) on February 14, 2019.
ENDOTOXIN-INDUCED
PROSTAGLANDIN
RELEASE
tained at 0, 15, 30, 60, and 90 min. Arterial blood gas and volume of packed red cell (VPRC) measurements were made at 0 and 90 min. ~~oup 2 consisted of eight dogs given endotoxin. Each dog was observed during a control 30min period in which average pressures and CO were measured and blood samples for arterial blood gases, VPRC, and PG analysis were drawn. After the control period purified Escherichiu coli endotoxin (Difco Laboratories, Detroit), 1 mg/kg diluted in normal saline, was injected into the PA. Pressures were monitored continuously. Blood samples for prostaglandin levels and CO measurements were obtained at 15, 30, 60, and 90 min. Arterial blood gas and VPRC measurements were made in the 90-min sample. Group 3 consisted of six dogs given endotoxin and acetylsalicylic acid (ASA). Powdered ASA (Merck & Co.) was dissolved in 5-10 ml ethyl alcohol and diluted in 40-50 ml normal saline in three experiments. In the remaining three experiments smaller volumes of saline (20 ml) were used to avoid the small precipitation of ASA. One dog was given 47 mg/kg iv as a slow injection over 5 min before the endotoxin was injected. The second dog was given a 4-mg/min continuous infusion for 90 min beginning with the injection of endotoxin. The remaining four dogs were given 4 mg/kg iv as a bolus injection before endotoxin and 4 mg/min for 90 min as a continuous infusion beginning with the injection of endotoxin. Group 4 consisted of six dogs given endotoxin and indomethacin. Powdered indomethacin (Merck Sharp & Dohme) was dissolved in 5-10 ml phosphate buffer at pH 7.40 and diluted in normal saline. Each dog received l-2 mg/kg iv as a bolus injection into the PA before endotoxin, followed by a 90-min continuous infusion at a rate of 1 mg/min beginning with the injection of endo toxin. Hemodynamic measurements and blood samples for arterial blood gases, VPRC, and prostaglandin analysis in the group 3 and a animals were obtained according to the schedule for group 2. Sequential changes in PGE and PGF for each series of samples were examined for statistical significance by analysis of variance and the Newman-Keuls sequential multiplecomparison procedure (27). The assay values were assumed to be lognormally distributed. However, for the purpose of illustration the data are expressed in terms of the mean value of the raw data & standard error (SE).
PGE mpg/ml 2
T
G 4
Endotoxin 0 Control l
Aorta
T
15 Port0 I Vein
30
60
90
30
60
9’0
3 2 I f 01
I I5
1 c
77
T
Renal Vein
6543” 2I O-
I5
PGF mkghl 3 t
o1
3b Time (min 1
60
Aorta a Endotoxin 0 Control
B T
t
1
C
15
1
60
Port01 Vein
I
$0
T
RESULTS
A comparison of PGE and PGF levels between five control dogs and eight dogs given endotoxin is shown in Fig. 1, A and B. In the control dogs there was no significant variation between the means of any 90-min sequence of samples for either PGE or PGF, regardless of sampling site. In the dogs given endotoxin, PGE levels in the aorta did not change, whereas PGF levels tended to rise slightly but did not reach statistical significance. The PGE and PGF levels in the portal vein were both significantly elevated at 15 min; thereafter, PGE levels tended to decrease toward normal, whereas PGF levels remained elevated. The PGE
0 1
t.---+ c
6T
----
I5 Renal Vein
+ -~-
30
TI
I
60
90
FIG. 1. Comparison of sequential changes in PGE (A) and PGF (B) between 5 contro1 dogs (open circles) and 8 dogs given endotoxin (closed circles). Samples were taken simultaneously from aorta, portal vein, and renal vein. Asterisk indicates a significant difference (P < 0.05) between corresponding value and control (C) vaIue,
Downloaded from www.physiology.org/journal/ajplegacy at Washington Univ (128.252.067.066) on February 14, 2019.
T
412
ANDERSON,
and PGF levels in the renal vein were significantly elevated 60 and 90 min after endotoxin. A comparison of PGE and PGF levels between the control dogs and dogs given endotoxin after either ASA or indomethacin pretreatment is shown in Fig. 2, A and B. In both PGE mpg/ml
l
ASA
IM 0 Control
I Portat
Vein
Rend
Vein
1
2 I 0I
d
i0
$0
9b
Time (mid PGF m&g/ml
IJ
Aorta
2 I 0i
Portd
3
TSAGARIS,
AND
KUIDA
groups PGE and PGF levels remained unchanged in the aorta, portal vein, and renal vein after endotoxin. The hemodynamic data for each of the four groups are shown in Table 1. In the control dogs, aortic pressure (AoP) and CO tended to fall gradually with time, whereas pulmonary artery pressure (PAP) and portal vein pressure (PVP) remained constant. In the dogs given endotoxin, there was an immediate rise in PAP and PVP with a corresponding fall in AoP. Aortic pressure, PAP, and PVP returned to normal, but there was a subsequent steady decrease in AoP. Cardiac output was not measured during the early hypotensive period after endotoxin, but at 15, 30, 60, and 90 min it was decreased from control levels. In the dogs pretreated with ASA, the hemodynamic effects of endotoxin were similar to the untreated dogs given endotoxin. In the dogs pretreated with indomethacin, there was an early rise in PAP and PVP with a fall in AoP and CO. Thereafter AoP, PVP, and PAP remained near control levels, whereas CO returned to control level and then decreased again at 90 min. The blood gas data and VPRC values for each of the four groups are shown in Table 2. In the control dogs PCQ and VPRC decreased over 90 min whereas Peg and pH remained constant. In the dogs given endotoxin PCO~ and pH both decreased whereas Paz remained constant and VPRC increased. In the ASA- and indomethacin-pretreated dogs Pox was higher both before and after endotoxin than in the other groups. In the ASA group Pcoz was decreased and fell further after endotoxin whereas pH was increased before and markedly decreased after endotoxin. In the indomethatin group PCO~ did not change and pH decreased. In the ASA group the rise in VPRC was comparable to that of the endotoxin group whereas in the indomethacin group there was only a slight rise in VPRC after endotoxin.
A
A
Aorta
JUBIZ,
Vein
2 I 0t Renal Vein
2
DISCUSSION
I 0
30
60
The results of this study indicate that PGE and PGF are released from the kidney and mesenteric organs during endotoxin shock in dogs. The validity of these data is dependent on the adequacy of the shock model employed. In this regard the hemodynamic alterations and changes in blood gas composition in the endotoxin-treated dogs correspond to those reported by others (10, 13, 19, 34). A bi-
90
Time (mid FIG. 2. Comparison of sequential changes in PGE (A) and PGF (23) between 5 control dogs, 6 dogs given endotoxin after acetylsalicylic acid (ASA) pretreatment, and 6 dogs given endotoxin after indomethacin (IM) pretreatment.
1. comparison
TABLE
2 z CJ
-
of corresponding Aortic
Time,
mmHg
5
15
30
60
90
125 11
120 9
108 10
107 12
Pressure,
Pulmonary
Artery =Hg
c
1s
30
60
90 _-~.-
12 1
12 1
12 1
11 1
6 1
min
c _~_
Pressure,
hemodynamic data between groufis J-4 ----
5
.-- .-
14
PortaI C
Vein
5
Pressure,
15
30
SE
137 8
2
z SE
106 30 64
66 8
72 8
55 4
47 6
10 15 152212
12
12
10
10
62188 1 111
3
2 SE
123 8
73 15
97 13
96 11
82 11
62 9
18 2
29 7
27 7
21 5
17 5
8 1
20 8 211
7
4
2 SE
122 71 615
99 7
118 9
122 8
118 8
17 17 11
7 1
16 9 211
8
I
2
Group 1: control methacin-pretreated
dogs. Group dogs given
1
26 5
14 16 12
2: dogs given endotoxin, endotoxin. Control period
17 17 11
55 11
Group 3: acetylsalicyk acid-pretreated = C, In groups 2-4 hemodynamic data
mmHg
60
Cardiac
90
c
4 11
4
111 10
7 11
6
8 13
Output,
15
per kg
30
60
90
85 13
79 14
92 20
59 13
80 9
55 8
47 5
51 6
45 8
9
63 3
34 7
36 9
38 9
31 7
10 10 12
67 7
43 7
62 11
70 14
56 8
dogs given at 5 min after
5
ml/min
endotoxin. endotoxin
Downloaded from www.physiology.org/journal/ajplegacy at Washington Univ (128.252.067.066) on February 14, 2019.
Group 4: indoare also shown.
ENDUTUXIN-INDUCED TABLE
uuhme
2.
PIIOSTAGLANDIN
&m/wison of corresfionding blood gas d&i red cells befz.oeengrau;bs l-4 _,_.._. .~~._ - “~_- ~-----I_-.- -~-I-
of packed
~I_..
--
Group Statistic c I
POQ, mmHg
mmHg PH y0
Groups
I
same
as in Table
413
and - - -~-~_
-
Gmtp3 I
I 90 min
90
C I
69 70 80 5 7 5 35 27 23 3 4 5 7.33 7.36 7.44 0.02 0.04 0.05 47 44 42 3 1 3
pco2,
VPRC,
KELEASE
min
c
-l-
78 91 3 5 18 25 2 4 7.24 7.39 0.03 0.06 51 47 6 3
-
90
C
min
I-----I90
-.--
91 11 I8 22 30 4 1 7.16 7.32 0.04 0.02 45 l55 '4 1
,
90
min
--
105
15 30 3 7.26 0.03 48
1
.-
1.
phasic fall in arterial pressure and rise in PVP and PAP are characteristic of this response. However, the untreated control dogs also developed hypotension with a decrease in VPRC. These findings are consistent with hypovolemia and are probably due to hemorrhage related to anticoagulation and numerous blood samples for radioimmunoassay. Our assumption that these dogs served as adequate controls for the endotoxin-treated dogs is based on the finding that PG levels did not change despite significant hemodynamic deterioration and that al1 dogs in each of the three groups were subject to a comparable degree of stress. In the pretreated dogs CO was decreased; we have no explanation for this, although anesthesia and regulated rcspiration may have played a role. Acetylsalicylic acid seemed to have negligible protective esect in the doses used but indomethacin appeared to sustain AoP and CO during the delayed phase of endotoxin shock, This contrasts with studies in which considerably larger doses of indomcthacin or ASA supported systemic arterial pressure during the delayed phase of endotoxin shock, provided inhibition of hepatic vein constriction, or improved survival (3, 7, 12, 14, 24, 28). Controlled respiration using 20 % 02 and 3 %I COG was employed because of anesthesi a-ind uced hv poxernia and data indicate respirator-induced hypocapnia. Our control that arterial Paz was maintained at normal levels, but PCQ was reduced in all groups. Endotoxin administration resulted in no change in Paz but caused a further decrease in Pcoz , indicating that regulated respiration did not completely prevent hyperventilation induced by shock and/or ASA. Arterial pH decreased markedly after endotoxin consistent with the metabolic acidosis characteristic of this increased VPRC indicated hemoconcentration response. (I). Indomethacin and ASA appeared to have little protective effect in terms of acidosis, although hemoconcentration appeared to be less marked in the indomethacinpretreated dogs. The release of PGE and PGF from the kidney and mescn-
teric organs during endotoxin shock in dogs may be accounted for by either increased synthesis or stimulation of the release of preformed prostaglandins. Since the release of PGE and PGF was significantly modified by indomethatin and ASA, known inhibitors of PG synthesis (8, 31), we feel the former possibility is more likely. The factors that influence PGE and PGF synthesis are incompletely understood. Ischernia, serotonin, bradykinin, catecholamines, and sympathetic nerve stimulation have been implicated (4, 9, 15, 20, 2 1). Thus, the time course of PGE and PGF release from the kidney and mesenteric organs may reflect the increased concentration of another vasoactivc hormone within the circulation during endotoxin shock, increased sympathetic activity, and/or tissue ischemia. The failure to demonstrate changes in PGE and PGF levels in aortic samples probably reflects the ability of the lung to eflectively metabolize these substances (25). Inactivating enzymes may be present within the liver as well (9). Thus, PGE and PGF released from the kidney and mesenteric organs probably have limited vasomotor potential in the systemic and hepatic circulation. The extent to which PGE and PGF influence local vasomotor tone and blood flow in the kidney and mesenteric organs before and during cndotoxin shock is difficult to determine from our data. In dogs, exogenously administered PGEl or PGEZ decreases vascular resistance and increases blood flow in femoral, brachial, mesenteric, splenic, and renal arteries, whereas PGFZ, increases vascular resistance and decreases blood flow in these vessels (5, 6, 23, 26). The possibility that increased release of PGE and PGF reflects their influence on local vasomotor tone cannot be proven from our data. Either vasodepressor or vasopressor effects could prevail since both PGE and PGF are released in a similar time course from the kidney and mesenteric organs. Inhibition of the delayed hemodynamic alterations by indomethacin corresponding to inhibition of PGE and PGF release suggests their possible role in this regard. A direct pressor effect of indomethacin on systemic arterioles cannot be excluded, however, since aortic pressure was maintained at a higher level in this group than in the other three. In contrast, the dose of ASA given failed to block the hemodynamic effects produced by endotoxin but did inhibit PGE and PGF release. It is possible, therefore, that ASA and indomethacin block the hemodynamic effects of endotoxin through inhibition of the vasomotor activity of other substances such as histamine, serotonin, catecholamines, and bradykinin (22, 29, 30). The greatly This and by
technical assistance of Mr. Don Anton and Mrs. Elena Rim is appreciated. study was supported by Public Health Service Grant HE-07618 the Utah Heart Association.
Received
for publication
7 January
1974.
REFERENCES 1. CHIEN, S., li. J. DELLENBACK, U. SHUNICHI, AND PIT. I. GREGERSEN. Hematocrit changes in endotoxin shock, Proc. $06. fiq?tZ. Viol. Med. 118: 1182-l 187, 1965. 2. COLLIER, J. G., A. G. HERMAN, AND J. R. VANE. Appearance of prostaglandins in the renal venous blood of dogs in response to
acute
systemic
produced by bleeding or endotoxin. 19p, 1973. 3. CULP, J. R., E. G. ERD~S, L. B. HINSHAW, AND D. D. HOLMES. Eflects of anti-inflammatory drugs in shock caused by injection of living E. coli cells. Proc. Sot. E+. Bid. Med. 137 : 2 19-223, 197 1. J. Physiol.,
London
hypotension 230:
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414
ANDERSON,
4. DAVIES, B. N., E. W. HORTON, AND B. G. WTHRINGTON. The occurrence of prostaglandin IX2 in splenic venous blood of the dog following splenic nerve stimulation. &it. J. Pharmacol. 32 : 12 7-l 35, 1968. 5. DAVIES, B. N., AND P. G. WITHRXNGTON. The effects of prostaglandin El, and E2 on the smooth muscle of the dog spleen and on its responses to catecholamines, angiotensin, and nerve stimulation. &it. J. Pharmacoi. 32 : I 36- 144, 1968. 6. DAVIES, B. N., ANI> P. G. WITHRXNGTON. Actions of prostaglandin Fza on the splenic vascular and capsular smooth muscle in the dog. Brit. J. Pharmacol. 4 I : 1-7, 7. ERD~S, E. G., L. B. HINSHAW,
methacin in endotoxin shock Med. 125: 916-919, 1967.
197 I. AND
C.
C.
GILL. Proc.
Effect
21.
of indo22.
8. FERREIRA, S. H., S. MONCADA, AND J. R. VANE. Indomethacin and aspirin abolish prostaglandin release from the spleen. Nature New Biol. 231: 237-239, 1971. 9. FERREIRA, S. H., AND J. R. VANE, Prostaglandins; their disappearance from and release into the circulation. iV&ture 216 : 868873, 1967. 10. GILBERT, R. P. Hemodynamic effects of endotoxin physiol. Rev. 40: 245-279, 1960. 11. HALL, R. C., AND R. L. HODQE. Vasoactive hormones in endotoxin shock: a comparative study in cats and dogs. J. Physiol., London 213: 69-84, 1971. 12. HALL, R. C., R. L. HODGE, R. IRVINE, F. KATIC, AND J. M. MIDDLETON. The effect of aspirin on the response to endotoxin.
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dog.
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in the
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R. M., T, G. BARILA, J. W. BURNS, AND H. P. MOCK. 13. HARDAWAY, Studies on pH changes in endotoxin and hemorrhagic shock. J. Surg. Res. 1: 278-284, 1961, L. B., L. A. SOLOMON, E. G. ERD~S, D. A, REINS, AND 14. HINSHAW, B. J. GUNTER. Effects of acetylsalicylic acid on the canine response to endotoxin. J. Pharmacol. Exftl. Therap. 157 : 665-67 I., 1967. S. W. The spontaneous release of prostaglandins into 15. HOLMES, the cerebral ventricles of the dog and the effect of external factors on this release. &it. J. Pharmacol. 38: 653-658, 1970. C. CHILD, AND IX. BARTHOLOMEW. Physio16. JUBIZ, W., J, FRAILEY, logic role of prostaglandins of the E (PGE), F (PGF) and AB (PGAB) groups. Estimation by radioimmunoassay in unextracted human plasma. Prostaglandins 2 : 471-489, 1972. IX., R. C. HUGHES, E. N. BENNETT, AND S. M. NADELA. x7, KESSLER, Evidence for the presence of prostaglandin-like material in the plasma of dogs with endotoxin shock. J. Lab. Clin. Med. 81: 85-94, 18. KOLOB,
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W, KATZ,
AND
A. P. THAT,,
Chemical
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25. 26.
27. 28.
29. 30.
31.
32, 33.
34.
JUBIZ,
TSAGARIS,
AND
KUIDA
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Downloaded from www.physiology.org/journal/ajplegacy at Washington Univ (128.252.067.066) on February 14, 2019.
Vol.
JOURNAL
228, No.
OF
2, February
PHYSIOLOGY
1975.
Printed
in U.S.A.
Endotoxin-induced
prostaglandin
E and F
release in dogs FRED L. ANDERSON, WILLIAM JUBIZ, THEOFILOS AND HIROSHI KUIDA Cardiovascdar and Metabolism Divisions, Veterans Administration College of bfedicine, Salt Lake City, Utah 84112
shock;
mesenteric
organs;
portal
vein;
renal
acid; indomethacin
SEVERAL VASOACTIVE SUBSTANCES have been implicated in the mechanism of canine endotoxin shock. These include histamine, serotonin, catecholamines, and bradykinin (11, 18, 32, 33). Prostaglandins E and F (PGE and PGF) are vasodepressor and vasopressor lipids, respectively, that are present in many canine tissues. Prostaglandin-like material has been identified in the plasma of dogs with endotoxin shock and acute systemic hypotension but specific identification of these substances has not been made (2, 17). The purpose of this study was to determine PGE and PGF levels by radioimmunoassay in sequential blood samples from the renal and portal veins and aorta during endotoxin shock in dogs. The influence of acetylsalicylic acid and indomethacin pretreatment on endotoxin-induced PGE and PGF reIease was also studied. An analysis of these experiments forms the basis for this report.
l
METHODS
Adult thetized
Hospital, and University of Utah
to avoid anesthesia-induced hypoxemia the animals were ventilated at a constant pressure and rate throughout the experiment with a positive-pressure respirator (Medtronics, Minneapolis) with a gas mixture of either room air (group I) or 20 % 02, 3 % CO2 , and 77 % nitrogen (groups 2, 3, and 4). Two catheters were introduced into the external jugular vein and advanced under fluoroscopy to either the left or right renal vein (RV) and pulmonary, artery (PA), respectively. In 20 of 25 dogs the left renal vein was catheterized. In these dogs the catheter tip was advanced deep into the renal pelvis to avoid contamination with spermatic vein or inferior vena cava blood. A third catheter was introduced into the carotid artery and advanced to the ascending aorta (Ao) for the purpose of sampling blood in as close proximity to the lungs as possible. The abdomen was then entered via a midline incision A small polyethylene cannula was inserted into a branch of the splenic vein and advanced to the portal vein (PV). Correct position of the RV catheter tip in the renal pelvis was confirmed by palpation. A second cannula was inserted into the femoral artery (FA) for the purpose of measuring arterial pressure, All catheters and cannulas were connected to Statham P23Db pressure transducers. Heparin, 10,000 U, was given intravenously to prevent clot formation in the catheters and can&as. Cardiac output (CO) was determined by the dye-dilution method. Indocyanine green dye (5-l 0 mg) was injected into the PA and sampled from the FA through a densitometer (Gilson Medical Electronics, Madison, Wis.) using a constant-rate withdrawal pump (Harvard Apparatus Co., Dover, Mass.). Pressures and the dye curves were recorded on an oscillographic recorder (Minneapolis Honeywell, Denver) Body temperature was not measured or controlled in any of the experimen ts. Blood samples for prostaglandin analysis were handled in the following manner. Each blood sample was immediately transferred to sterile glass tubes containing 0.3 ml of 20 % sodium citrate. After centrifugation for 15 min the plasma was transferred to 5-ml glass containers and frozen immediately. Each sample was analyzed for PGE and PGF by the radioimmunoassay method of Jubiz et al. (16). The antisera used in the present studies were the same that we utilized in that study. Four groups of animals were studied. Group I consisted of five control dogs in which no endotoxin was given. Pressures were recorded continuously for 90 min. Blood samples for prostaglandin levels and CO measurements were ob-
ANDERSON, FRED L., WILLIAM JUBIZ, THEOFKOS J. TSACARIS, HIROSHI KUIDA. Endotoxin-induced ~rostuglandin E and F release in dogs. Am. J. Physiol. 228(Z): 410-414. 1975.-Prostaglandin E and F (PGE and PGF) 1evels in sequential blood samples obtained simultaneously from the renal and portal veins and aorta during endotoxin shock in dogs were determined by radioimmunoassay. Four groups of dogs were studied. In five control dogs in which no endotoxin was given, PGE and PGF levels did not change significantly at 0, 15, 30, 60, and 90 min. In eight dogs given endotoxin alone, PGE and PGF levels did not change in the aorta. In samples taken from the portal vein there was a significant rise in PGE and PGF 15 min after endotoxin, whereas renal vein PGE and PGF did not become significantly elevated until 60 and 90 min after endotoxin. In six dogs pretreated with acetylsalicylic acid and six dogs pretreated with indomethacin, PGE and PGF levels did not change after endotoxin. Indomethacin modified the delayed hemodynamic effects of endotoxin whereas acetylsalicylic acid did not. Neither drug blocked the immediate hemodynamic effects of endotoxin. Endotoxin-induced PGE and PGF release is probably due to increased synthesis. The mechanism whereby synthesis is stimulated and the extent to which vasomotor tone is influenced by PGE and PGF during endotoxin shock cannot be determined from our data. AND
radioimmunoassay; vein; acetylsalicylic
J. TSAGARIS,
mongrel dogs (average weight 23 kg) were aneswith sodium pentobarbita1 (30 mg/kg iv). In order 410
Downloaded from www.physiology.org/journal/ajplegacy at Washington Univ (128.252.067.066) on February 14, 2019.
ENDOTOXIN-INDUCED
PROSTAGLANDIN
RELEASE
tained at 0, 15, 30, 60, and 90 min. Arterial blood gas and volume of packed red cell (VPRC) measurements were made at 0 and 90 min. ~~oup 2 consisted of eight dogs given endotoxin. Each dog was observed during a control 30min period in which average pressures and CO were measured and blood samples for arterial blood gases, VPRC, and PG analysis were drawn. After the control period purified Escherichiu coli endotoxin (Difco Laboratories, Detroit), 1 mg/kg diluted in normal saline, was injected into the PA. Pressures were monitored continuously. Blood samples for prostaglandin levels and CO measurements were obtained at 15, 30, 60, and 90 min. Arterial blood gas and VPRC measurements were made in the 90-min sample. Group 3 consisted of six dogs given endotoxin and acetylsalicylic acid (ASA). Powdered ASA (Merck & Co.) was dissolved in 5-10 ml ethyl alcohol and diluted in 40-50 ml normal saline in three experiments. In the remaining three experiments smaller volumes of saline (20 ml) were used to avoid the small precipitation of ASA. One dog was given 47 mg/kg iv as a slow injection over 5 min before the endotoxin was injected. The second dog was given a 4-mg/min continuous infusion for 90 min beginning with the injection of endotoxin. The remaining four dogs were given 4 mg/kg iv as a bolus injection before endotoxin and 4 mg/min for 90 min as a continuous infusion beginning with the injection of endotoxin. Group 4 consisted of six dogs given endotoxin and indomethacin. Powdered indomethacin (Merck Sharp & Dohme) was dissolved in 5-10 ml phosphate buffer at pH 7.40 and diluted in normal saline. Each dog received l-2 mg/kg iv as a bolus injection into the PA before endotoxin, followed by a 90-min continuous infusion at a rate of 1 mg/min beginning with the injection of endo toxin. Hemodynamic measurements and blood samples for arterial blood gases, VPRC, and prostaglandin analysis in the group 3 and a animals were obtained according to the schedule for group 2. Sequential changes in PGE and PGF for each series of samples were examined for statistical significance by analysis of variance and the Newman-Keuls sequential multiplecomparison procedure (27). The assay values were assumed to be lognormally distributed. However, for the purpose of illustration the data are expressed in terms of the mean value of the raw data & standard error (SE).
PGE mpg/ml 2
T
G 4
Endotoxin 0 Control l
Aorta
T
15 Port0 I Vein
30
60
90
30
60
9’0
3 2 I f 01
I I5
1 c
77
T
Renal Vein
6543” 2I O-
I5
PGF mkghl 3 t
o1
3b Time (min 1
60
Aorta a Endotoxin 0 Control
B T
t
1
C
15
1
60
Port01 Vein
I
$0
T
RESULTS
A comparison of PGE and PGF levels between five control dogs and eight dogs given endotoxin is shown in Fig. 1, A and B. In the control dogs there was no significant variation between the means of any 90-min sequence of samples for either PGE or PGF, regardless of sampling site. In the dogs given endotoxin, PGE levels in the aorta did not change, whereas PGF levels tended to rise slightly but did not reach statistical significance. The PGE and PGF levels in the portal vein were both significantly elevated at 15 min; thereafter, PGE levels tended to decrease toward normal, whereas PGF levels remained elevated. The PGE
0 1
t.---+ c
6T
----
I5 Renal Vein
+ -~-
30
TI
I
60
90
FIG. 1. Comparison of sequential changes in PGE (A) and PGF (B) between 5 contro1 dogs (open circles) and 8 dogs given endotoxin (closed circles). Samples were taken simultaneously from aorta, portal vein, and renal vein. Asterisk indicates a significant difference (P < 0.05) between corresponding value and control (C) vaIue,
Downloaded from www.physiology.org/journal/ajplegacy at Washington Univ (128.252.067.066) on February 14, 2019.
T
412
ANDERSON,
and PGF levels in the renal vein were significantly elevated 60 and 90 min after endotoxin. A comparison of PGE and PGF levels between the control dogs and dogs given endotoxin after either ASA or indomethacin pretreatment is shown in Fig. 2, A and B. In both PGE mpg/ml
l
ASA
IM 0 Control
I Portat
Vein
Rend
Vein
1
2 I 0I
d
i0
$0
9b
Time (mid PGF m&g/ml
IJ
Aorta
2 I 0i
Portd
3
TSAGARIS,
AND
KUIDA
groups PGE and PGF levels remained unchanged in the aorta, portal vein, and renal vein after endotoxin. The hemodynamic data for each of the four groups are shown in Table 1. In the control dogs, aortic pressure (AoP) and CO tended to fall gradually with time, whereas pulmonary artery pressure (PAP) and portal vein pressure (PVP) remained constant. In the dogs given endotoxin, there was an immediate rise in PAP and PVP with a corresponding fall in AoP. Aortic pressure, PAP, and PVP returned to normal, but there was a subsequent steady decrease in AoP. Cardiac output was not measured during the early hypotensive period after endotoxin, but at 15, 30, 60, and 90 min it was decreased from control levels. In the dogs pretreated with ASA, the hemodynamic effects of endotoxin were similar to the untreated dogs given endotoxin. In the dogs pretreated with indomethacin, there was an early rise in PAP and PVP with a fall in AoP and CO. Thereafter AoP, PVP, and PAP remained near control levels, whereas CO returned to control level and then decreased again at 90 min. The blood gas data and VPRC values for each of the four groups are shown in Table 2. In the control dogs PCQ and VPRC decreased over 90 min whereas Peg and pH remained constant. In the dogs given endotoxin PCO~ and pH both decreased whereas Paz remained constant and VPRC increased. In the ASA- and indomethacin-pretreated dogs Pox was higher both before and after endotoxin than in the other groups. In the ASA group Pcoz was decreased and fell further after endotoxin whereas pH was increased before and markedly decreased after endotoxin. In the indomethatin group PCO~ did not change and pH decreased. In the ASA group the rise in VPRC was comparable to that of the endotoxin group whereas in the indomethacin group there was only a slight rise in VPRC after endotoxin.
A
A
Aorta
JUBIZ,
Vein
2 I 0t Renal Vein
2
DISCUSSION
I 0
30
60
The results of this study indicate that PGE and PGF are released from the kidney and mesenteric organs during endotoxin shock in dogs. The validity of these data is dependent on the adequacy of the shock model employed. In this regard the hemodynamic alterations and changes in blood gas composition in the endotoxin-treated dogs correspond to those reported by others (10, 13, 19, 34). A bi-
90
Time (mid FIG. 2. Comparison of sequential changes in PGE (A) and PGF (23) between 5 control dogs, 6 dogs given endotoxin after acetylsalicylic acid (ASA) pretreatment, and 6 dogs given endotoxin after indomethacin (IM) pretreatment.
1. comparison
TABLE
2 z CJ
-
of corresponding Aortic
Time,
mmHg
5
15
30
60
90
125 11
120 9
108 10
107 12
Pressure,
Pulmonary
Artery =Hg
c
1s
30
60
90 _-~.-
12 1
12 1
12 1
11 1
6 1
min
c _~_
Pressure,
hemodynamic data between groufis J-4 ----
5
.-- .-
14
PortaI C
Vein
5
Pressure,
15
30
SE
137 8
2
z SE
106 30 64
66 8
72 8
55 4
47 6
10 15 152212
12
12
10
10
62188 1 111
3
2 SE
123 8
73 15
97 13
96 11
82 11
62 9
18 2
29 7
27 7
21 5
17 5
8 1
20 8 211
7
4
2 SE
122 71 615
99 7
118 9
122 8
118 8
17 17 11
7 1
16 9 211
8
I
2
Group 1: control methacin-pretreated
dogs. Group dogs given
1
26 5
14 16 12
2: dogs given endotoxin, endotoxin. Control period
17 17 11
55 11
Group 3: acetylsalicyk acid-pretreated = C, In groups 2-4 hemodynamic data
mmHg
60
Cardiac
90
c
4 11
4
111 10
7 11
6
8 13
Output,
15
per kg
30
60
90
85 13
79 14
92 20
59 13
80 9
55 8
47 5
51 6
45 8
9
63 3
34 7
36 9
38 9
31 7
10 10 12
67 7
43 7
62 11
70 14
56 8
dogs given at 5 min after
5
ml/min
endotoxin. endotoxin
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Group 4: indoare also shown.
ENDUTUXIN-INDUCED TABLE
uuhme
2.
PIIOSTAGLANDIN
&m/wison of corresfionding blood gas d&i red cells befz.oeengrau;bs l-4 _,_.._. .~~._ - “~_- ~-----I_-.- -~-I-
of packed
~I_..
--
Group Statistic c I
POQ, mmHg
mmHg PH y0
Groups
I
same
as in Table
413
and - - -~-~_
-
Gmtp3 I
I 90 min
90
C I
69 70 80 5 7 5 35 27 23 3 4 5 7.33 7.36 7.44 0.02 0.04 0.05 47 44 42 3 1 3
pco2,
VPRC,
KELEASE
min
c
-l-
78 91 3 5 18 25 2 4 7.24 7.39 0.03 0.06 51 47 6 3
-
90
C
min
I-----I90
-.--
91 11 I8 22 30 4 1 7.16 7.32 0.04 0.02 45 l55 '4 1
,
90
min
--
105
15 30 3 7.26 0.03 48
1
.-
1.
phasic fall in arterial pressure and rise in PVP and PAP are characteristic of this response. However, the untreated control dogs also developed hypotension with a decrease in VPRC. These findings are consistent with hypovolemia and are probably due to hemorrhage related to anticoagulation and numerous blood samples for radioimmunoassay. Our assumption that these dogs served as adequate controls for the endotoxin-treated dogs is based on the finding that PG levels did not change despite significant hemodynamic deterioration and that al1 dogs in each of the three groups were subject to a comparable degree of stress. In the pretreated dogs CO was decreased; we have no explanation for this, although anesthesia and regulated rcspiration may have played a role. Acetylsalicylic acid seemed to have negligible protective esect in the doses used but indomethacin appeared to sustain AoP and CO during the delayed phase of endotoxin shock, This contrasts with studies in which considerably larger doses of indomcthacin or ASA supported systemic arterial pressure during the delayed phase of endotoxin shock, provided inhibition of hepatic vein constriction, or improved survival (3, 7, 12, 14, 24, 28). Controlled respiration using 20 % 02 and 3 %I COG was employed because of anesthesi a-ind uced hv poxernia and data indicate respirator-induced hypocapnia. Our control that arterial Paz was maintained at normal levels, but PCQ was reduced in all groups. Endotoxin administration resulted in no change in Paz but caused a further decrease in Pcoz , indicating that regulated respiration did not completely prevent hyperventilation induced by shock and/or ASA. Arterial pH decreased markedly after endotoxin consistent with the metabolic acidosis characteristic of this increased VPRC indicated hemoconcentration response. (I). Indomethacin and ASA appeared to have little protective effect in terms of acidosis, although hemoconcentration appeared to be less marked in the indomethacinpretreated dogs. The release of PGE and PGF from the kidney and mescn-
teric organs during endotoxin shock in dogs may be accounted for by either increased synthesis or stimulation of the release of preformed prostaglandins. Since the release of PGE and PGF was significantly modified by indomethatin and ASA, known inhibitors of PG synthesis (8, 31), we feel the former possibility is more likely. The factors that influence PGE and PGF synthesis are incompletely understood. Ischernia, serotonin, bradykinin, catecholamines, and sympathetic nerve stimulation have been implicated (4, 9, 15, 20, 2 1). Thus, the time course of PGE and PGF release from the kidney and mesenteric organs may reflect the increased concentration of another vasoactivc hormone within the circulation during endotoxin shock, increased sympathetic activity, and/or tissue ischemia. The failure to demonstrate changes in PGE and PGF levels in aortic samples probably reflects the ability of the lung to eflectively metabolize these substances (25). Inactivating enzymes may be present within the liver as well (9). Thus, PGE and PGF released from the kidney and mesenteric organs probably have limited vasomotor potential in the systemic and hepatic circulation. The extent to which PGE and PGF influence local vasomotor tone and blood flow in the kidney and mesenteric organs before and during cndotoxin shock is difficult to determine from our data. In dogs, exogenously administered PGEl or PGEZ decreases vascular resistance and increases blood flow in femoral, brachial, mesenteric, splenic, and renal arteries, whereas PGFZ, increases vascular resistance and decreases blood flow in these vessels (5, 6, 23, 26). The possibility that increased release of PGE and PGF reflects their influence on local vasomotor tone cannot be proven from our data. Either vasodepressor or vasopressor effects could prevail since both PGE and PGF are released in a similar time course from the kidney and mesenteric organs. Inhibition of the delayed hemodynamic alterations by indomethacin corresponding to inhibition of PGE and PGF release suggests their possible role in this regard. A direct pressor effect of indomethacin on systemic arterioles cannot be excluded, however, since aortic pressure was maintained at a higher level in this group than in the other three. In contrast, the dose of ASA given failed to block the hemodynamic effects produced by endotoxin but did inhibit PGE and PGF release. It is possible, therefore, that ASA and indomethacin block the hemodynamic effects of endotoxin through inhibition of the vasomotor activity of other substances such as histamine, serotonin, catecholamines, and bradykinin (22, 29, 30). The greatly This and by
technical assistance of Mr. Don Anton and Mrs. Elena Rim is appreciated. study was supported by Public Health Service Grant HE-07618 the Utah Heart Association.
Received
for publication
7 January
1974.
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ANDERSON,
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