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Diminution of contractile response of the aorta from endotoxin-injected rats
European Journal of Pharmacology, 141 (1987) 117-122 Elsevier
117
EJP 00893
Diminution of contractile response of the aorta from endotoxin-injected rats I c h i r o W a k a b a y a s h i *, K a t s u h i k o H a t a k c 1, Eizo K a k i s h i t a a n d K i y o y a s u N a g a i Second Department of Internal Medicine and ! Department of Legal Medicine, Hyogo Collegeof Medicine, Hyogo, 663, Japan
Received 5 January 1987, revised MS received 10 April 1987, accepted 9 June 1987
The contractility of a helical strip of the thoracic aorta was studied in rats injected intraperitoneally with endotoxin. The contractile response to any of the agonistic agents, KC1, norepinephrine or 5-hydroxytryptamine was time dependently diminished in the endotoxin-injected rats compared to the controls. This diminution preceded the depression of blood pressure. When the external calcium concentration was increased from 2.5 to 7.5 mM after the KC1 (80 mM)-induced contractile response reached a plateau, the diminished contractile response was reversed in the endotoxin-injected group. The strips from the endotoxin-injected rats showed a higher 45CAC12uptake into the vascular tissue with the KCl-stimulated contraction. These findings suggest that the blood pressure depression during endotoxic shock may be attributed partially to the diminished contractility of the blood vessels and that this diminution is induced by a disorder of calcium utilization within vascular smooth muscle during vascular contraction. Endotoxin; Vasoconstriction; Calcium
1. Introduction Circulatory failure in sepsis exerts a serious effect on the prognosis and frequently does not respond to various types of therapeutic management. However, the precise mechanism of cardiovascular abnormality in endotoxic shock is still unknown. It has been reported recently that endotoxin treatment induces vascular hyporesponsiveness to catecholamines in vivo (Parratt, 1973; Auclair et al., 1982). On the other hand, in vitro evaluation of the vascular reactivity during endotoxic shock has so far been httle described. Pomerantz et al. (1982) reported that the contractile response to norepinephrine and prostaglandin endoperoxide was reduced in thoracic aorta removed from rats injected intravenously (i.v.) with endotoxin. The cause of this diminished contractile response in vitro is still unknown. Recent * To whom all correspondence should be addressed: Second Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-Cho, Nishinomiya, Hyogo, 663, Japan.
studies using cardiac preparations in vitro have demonstrated that endotoxin induces cardiac depression which is associated with alterations in calcium metabohsm in the heart (Hess et al., 1980; Parker and Adams, 1981). The purpose of this study was to investigate vasocontractility changes after endotoxin treatment. Thoracic aortas from rats were used for this purpose and also to examine the mechanism of the contractile changes associated with calcium metabolism. 2. Materials and methods 2.1, Administration of endotoxin
Male Wistar rats (350-450 g) under anesthesia with sodium pentobarbital (30 m g / k g ) were treated by intraperitoneal (i.p.) injection of 10 m g / k g of endotoxin (lipopolysaccharide B from E. coli 0 2 6 : B 6 Difco Laboratories) diluted in physiological saline. The controls were rats treated i.p. with the same volume of physiological saline.
0014-2999/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
118
A catheter was inserted into the left femoral artery of rats under anesthesia with i.p. sodium pentobarbital (30 mg/kg), and a pressure transducer (Stotham, Model 23Db) was connected to measure MAP.
tetraacetic acid). A scintillation mixture was added and the radioactivity was determined with a liquid scintillation counter (Packard Tri-Carb Model 460 Liquid scintillation spectrometer). The rate of Ca uptake was calculated as c.p.m./kg of the La-resistant 45Ca fraction divided by c.p.m./ /~mol Ca of the specific activity of 45Ca-containing medium.
2.3. Observation of isometric contraction ,
2.5. Substances
The rats were killed by a blow on the neck. The thoracic aorta was removed and immediately placed in Krebs-Ringer solution (mM: 118 NaC1, 4.7 KCI, 25 N a H C O 3, 1.2 KH2PO4, 1.2 MgSO 4 • 7H20, 2.5 CaC12 and 10 glucose; p H 7.4), and helical strip specimens of 2 × 15 mm were prepared; care was taken not to injure the intimal layer (Furchgott and Bhadrakom, 1953). Each specimen was carefully suspended between hooks in an organ chamber containing 10 ml of KrebsRinger solution with a gas stream (37 o C) of 95% 02-5% CO 2 and attached to a force transducer connected to a Nihon Kohden polygraph under a load of 1 g. After 1.5 h of stabilization, the isometric contraction was measured and expressed in terms of mg contractile force/rag wet tissue
The drugs used were: (-)-norepinephrine hydrochloride (Sigma Chemical), 5-hydroxytryptamine creatinine sulfate (Sigma Chemical) and 45CAC12 (New England Nuclear). The concentration of each drug was expressed as the final concentration in the bath.
2.2. Measurement of mean arterial blood pressure (MAP)
2.6. Statistics The results of the experiments are expressed as means + S.E.M. The maximum response (Emax) and ECs0, the concentration producing a halfmaximal response were determined graphically from individual dose-response curves. Statistical analyses were performed with Student's t-test and a P value less than 0.05 was taken as significant.
weight. 2.4. Measurement of tissue
45CaCl2 uptake
into vascular
A ring-shaped vascular strip 5 mm long was prepared from the aorta. After 1.5 h of stabilization in 5 ml of the above buffer not containing K H 2 P O 4 under a gas stream of 95% 02-5% CO2, the ratio of Ca uptake into the vascular strip was determined by means of the low temperature 3+ La method (Godfraind, 1976). The strip was treated with 0.4 /~Ci/ml of 45CAC12 together with the agonistic agent. After 5 min, the strip was taken out and immediately transferred into 2 ° C controlled 50 mM LaC13 solution (mM : 118 NaC1, 4.7 KC1, 1.2 MgC12, 10 glucose, 50 LaC13 and 15 Tris-maleate; pH 6.8). The wet weight of the strip was measured after a 60 min wash. The strip was then allowed to stand overnight in 2 ml of 20 mM E G T A (ethyleneglycol-bis-(2-aminoethylether)-
3. Results
3.1. Changes of mean arterial blood pressure after endotoxin administration Figure 1 shows the blood pressure changes before and after the i.p. injection of endotoxin. The blood pressure displayed no significant changes until 3 h after the endotoxin injection but fell to shock level at 6 h. Anesthesia with i.p. sodium pentobarbital had no effect on the blood pressure (data not shown). 3.2. Effect of endotoxin administration on aorta contractility Figure 2 shows the changes in the contractile responses induced by KC1, norepinephrine or 5hydroxytryptamine before and after the i.p. injec-
119 120
10mln$
B) Endotoxin
A) Control 100
o= t, 0.s
LL
8O E
o 60
40 KC~8OmM
20
,
,
0
15.
i 3
6
Time (hr)
Fig. 1. Time course of changes in mean arterial blood pressure (MAP) after intraperitoneal injection of 10 mg/kg endotoxin. Each point represents the mean_+ S.E. * Statistically significant (P < 0.05) differences compared to the values before endotoxin treatment, n = 8.
tion of endotoxin. The responses to each agonist diminished gradually after the endotoxin injection. The ECs0 values for each agonist with aortas from endotoxin-injected rats increased significantly from 1.5 h after injection of endotoxin (table 1). The involvement of calcium in the contractile response to KC1 was investigated next. The contractile response to 80 mM KC1 was markedly diminished in the endotoxin-injected group compared to the controls. However, an additional 5
KC~ 80raM
Fig. 3. Representative recordings of additional effect of 5 mM CaC12 on contractile response caused by 80 mM KCI. (A) Control, (B) 6 h after intraperitoneal injection of 10 m g / k g endotoxin. Vertical bars illustrate percentage enhancement of KC1 (80 mM)-induced contraction by addition of 5 mM CaCI 2. ([2) control, ([]) 6 h after endotoxin injection. * Statistically significant (P < 0.01) differences between responses in controls and endotoxin-injected animals.
mM CaC12 produced a marked enhancement of the contractility and the diminished contractile response to KC1 was reversed in the endotoxin-injected group (fig. 3).
3.3. Effect of endotoxin administration on calcium uptake of aorta The 5 min uptake of 45CAC12 into vascular tissue did not differ between the two groups during the static phase. However, upon stimulation with 80 mM KC1, the aorta strips from the endo-
A
C
!
B .........
~oo. °°S°°°°°°*°
.~
"
i ~
I~V~T
~. . . .
o--o ] h,s (n-s) olb-.-~ Sh,. In- s / ~ , , o . ~ , , ' *
~.°°
~...L--~
" ®
u.
...... KCJ~(raM)
/
..'.
...~ .............. 4
: ,::,, ,0-,.
,~-.
,;-.
,;-.
~orepim~nr,~ (M)
~'o-.
~0-.
5×1o'-;lo-',
s~lo-.
~-,
2xw"
5- Hydro~ytryptami~ (M)
Fig. 2. Effect of intraperitoneal injection of 10 m g / k g endotoxin on contractile responses upon exposure to cumulative additions of KCl (A), norepinephrine (B) or 5-hydroxytryptamine (C). Vertical lines indicate S.E. * Statistically significant (P < 0.05) differences between responses in controls and endotoxin-injected animals.
120 TABLE 1 EC50 values and maximum contractile effects of KCI, norepinephrine (NE) and 5-hydroxytryptamine (5-HT) in rat thoracic aorta before and after intraperitoneal injection of 10 m g / k g endotoxin. Values are means ___S.E. Drug
ECs0 (M) Before
1.5 h
3h
6h
KCI NE 5-HT
(2.34 :t: 0.23) × 10- 2 (6.215-0.24) x 10 -9 (1.96 + 0.16) × 10- s
(4.07 + 0.52) × 10- 2 . (2.08 +0.27) x 10 -8 * (3.49 5- 0.43) × 10 - s .
(5.51 + 0.54) × 10- 2 . (3.70+0.31)× 10 - s * (3.03 5- 0.31) × 10- s .
(5.86 + 0.48) × 10- 2 . (5.14+0.74) x 10 -8 * (4.05 5- 0.63) × 10 - s .
Drug
Maximum contraction (mg tension/mg wet tissue)
KC1 NE 5-HT
Before
1.5 h
3h
6h
166 _+10.6 215.1+12.6 202.4 5-12.8
139.5_+17.3 194.1_+18.5 175.4 + 10.3
82.65- 7.1 * 130.65- 9.8 * 97.8 _+14.2 *
80.35- 8.7 * 84.3-+11.4 * 79.3 __ 14.8 *
• Denotes significant difference between control and endotoxin-treated preparations (P < 0.05).
KC~
~,<~0.01 I l
p<0.0s I"-I
f
C~ control I ~ endotoxin
ba~l NS r~ 100
(A)
m
g~
0
(B)
Fig. 4. Effect of intraperitoneal injection of 10 m g / k g endotoxin on Ca uptake, 5 min after vehicle administration (basal) and 80 mM KC1 stimulation (KC1) (A), and isometric tension development, 5 rain after 80 mM KC1 stimulation (B). (0) control, ([]) 6 h after endotoxin injection. * P < 0.05, * * P < 0.01 compared to the values obtained before endotoxin treatment.
toxin-injected rats showed a higher 4~CaC12 uptake than the control tissues (fig. 4A). The 5 min contractile force after stimulation with 80 mM KCI was greatly diminished in the endotoxin-injected group (fig. 4B).
4. Discussion
The contractility of the aorta strips from endotoxin-treated rats was found to diminish gradually prior to the depression of blood pressure. This diminution of the contractile response can trigger circulatory failure in endotoxic shock. Seaman
and Greenway (1984) reported from an in vivo study on the contractility of the hepatic vein that the contractile response to nerve stimulation or infusion of norepinephrine or angiotensin II was diminished in the endotoxin-injected group. Auclair et al. (1986) suggested, on the basis of in vivo and in vitro studies, that endotoxin has a potent blocking action on vascular a-adrenoceptors. In the present in vitro study, the contractile response to norepinephrine and 5-hydroxytryptamine, which is thought to be composed of biphasic components, namely, a phasic component (mobilization of intracellular calcium) and a tonic component (influx of extracellular calcium due to opening of the calcium channel) (Godfraind and Kaba, 1972; Nakaki et al., 1985), was reduced and the KCl-induced contraction which is not mediated by receptor stimulation but depolarization of the cell membrane was also reduced in the endotoxin-injected group. These results suggest that the diminished vasocontractility caused by endotoxin treatment is due not only to abnormality of postsynaptic specific receptors but to alterations of the common contractile mechanism after stimulation of the receptors. Abnormality of the energy metabolism occurs during endotoxic shock, with a consequent inhibition of transmembranous ion migration (Schumer, 1984). The ATP-dependent calcium uptake in vascular smooth muscle, at the level of subcellular fractions, is damaged by the action of endotoxin
121
(Soulsby et al., 1976; Soulsby et al., 1980). Pomeranz et al. (1982) reported that the contractile response to norepinephrine was reduced in thoracic aortas excised from rats treated by i.v. injection of endotoxin and they speculated that this diminution of the contractile response resuited from the decreased calcium uptake by microsomes and mitochondria, which are the main storage pool of calcium. In the present experiments, KCl-induced contraction, which is dependent on extracellular calcium (Godfraind and Kaba, 1972), was also diminished in the endotoxin-injected groups. Thus, reduction of the intracellular calcium reserve is probably not the reason for the diminution of the contractile response. When the external calcium concentration was increased after the KC1 (80 mM)-induced contractile response had reached a plateau, the diminished contractile response was reversed in the endotoxin-injected groups. Instead, in contrast to the marked diminution of the 5 rain contractile force after stimulation with 80 mM KC1 in the endotoxin-injected group, the rate of 45CaC12 uptake during corresponding period was enhanced in this group compared to the controls. The 43CaC12 uptake stimulated by KC1 corresponds to the calcium influx through the calcium channel due to depolarization of the plasma membrane (Godfraind and Kaba, 1972). Accordingly, the voltagedependent calcium channel might not be damaged in the endotoxin-injected group. Ives et al. (1986) recently reported that the hypotension in endotoxin-treated rats was reversed by a calcium channel agonist, BAY k 8644. These findings therefore suggest that, in the endotoxin-injected group, disturbance of intracellular calcium utilization occurs in association with the excitation-contraction coupling of the vascular smooth muscle. One possible explanation for the decreased intracellular calcium utilization is an enhanced sequestration of calcium from cytosol in endotoxemia rather than diminished sequestration. Recent studies have demonstrated that the vascular endothelium plays an obligatory role in the regulation of vascular tonus (Furchgott, 1983). The endothelium-dependent relaxing response to a c e t y l c h o l i n e ( 1 0 - 6 M ) , which was expressed as a
percentage depression of the contractile responses to norepinephrine (10 -7 M), did not differ between control and endotoxin-injected groups (control: 55.3 + 3.1%, 6 h after endotoxin injection: 56.3 + 3.3%), and the vasocontraction caused by KC1, norepinephrine or 5-hydroxytryptamine was not altered by removal of the endothelium in both groups (data not shown). Therefore, the decreased vasocontractility in endotoxin-injected rat aortas is not due to changes in endothelial modulation of vascular tonus. The present experiments led to the conclusions that, in endotoxin-injected rats, reduction of aortic contractility is caused by a disorder of calcium utilization in association with the intracellular excitation-contraction coupling of vascular smooth muscles and that diminution of the contractile response of vascular smooth muscle can trigger circulatory failure in endotoxic shock.
Acknowledgement The authors wish to thank Dr. Kazuo Nagai for his valuable advice.
References Auclair, M.C., A. Carli and P. Lechat,~!982 , Decrease of the hypertensive responses to phenylephrine in the rat submitted to a sublethal dose of E. coli endotoxin. Restoration by indomethacin, J. Pharmacol. (Pads) 13, 341. Auclair, M.C., P. Svinareff and H. Schmitt, 1986, Vascular a-adrenoceptor blockade by E. coli endotoxin in the rat, European J. Pharmacol. 127, 121. Furchgott, R.F., 1983, Role of endothelium in response of vascular smooth muscle, Circ. Res. 53, 557. Furchgott, R.F. and S. Bhadrakom, 1953, Reactions of strips of rabbit aorta to epinephrine, isopropylarterenol, sodium nitrite and other drugs, J. Pharmacol. Exp. Ther. 108, 129. Godfraind, T., 1976, Calcium exchange in vascular smooth muscle, action of noradrenaline and lanthanum, J. Physiol. 260, 21. Godfraind, T. and A. Kaba, 1972, The role of calcium in the action of drugs on vascular smooth muscle, Axch. Int. Pharmacodyn. 196 (Suppl.), 35. Hess, M.L., S.M. Kreuse and P. Komwatana, 1980, Myocardial failure and excitation-contraction coupling in canine endotoxin shock: role of histamine and the sarcoplasmic reticulure, Circ. Shock 7, 277. Ives, N., J.W. King, B. Chernow and B.L. Roth, 1986, BAY k
122 8644, a calcium channel agonist, reverses hypotension in endotoxin-shocked rats, European J. Pharmacol. 130, 169. Nakaki, T., B.L. Roth, D. Chuang and E. Costa, 1985, Phasic and tonic components in 5-HT2, receptor-mediated rat aorta contraction: Participation of Ca ++ channels and phospholipase C 1, J. Pharmacol. Exp. Ther. 234, 442. Parker, J.L. and H.R. Adams, 1981, Contractile dysfunction of atrial myocardium from endotoxin-shocked guinea pigs, Am. J. Physiol. 240, H954. Parratt, J.R., 1973, Myocardial and circulatory effects of E. coli endotoxin; modification of responses to catecholamines, Br. J. Pharmacol. 47, 12. Pomerantz, K., L. Casey, J.R. Fletcher and P.W. RamweU, 1982, Vascular reactivity in endotoxin shock: Effect of lidocaine or indomethacin pretreatment, Adv. Shock Res. 7, 191.
Schumer, W., 1984, Subcellular response to septic shock, in: Clinical Surgery International, Vol. 9, (Churchill Livingstone) p. 61. Seaman, K.L. and C.V. Greenway, 1984, Loss of hepatic venous responsiveness after endotoxin in anesthetized cats, Am. J. Physiol. 246, H658. Soulsby, M.E., C.L: Bennett and M.L. Hess, 1980, Canine arterial calcium transport during endotoxin shock, Circ. Shock 7, 139. Soulsby, M.E., G.D. Ford, J.A. Davis, K. Wincklhofer and M.L. Hess, 1976, The effect of gram-negative endotoxin on calcium accumulation by subcellular fractions of vascular smooth muscle, Circ. Shock 3, 325.
117
EJP 00893
Diminution of contractile response of the aorta from endotoxin-injected rats I c h i r o W a k a b a y a s h i *, K a t s u h i k o H a t a k c 1, Eizo K a k i s h i t a a n d K i y o y a s u N a g a i Second Department of Internal Medicine and ! Department of Legal Medicine, Hyogo Collegeof Medicine, Hyogo, 663, Japan
Received 5 January 1987, revised MS received 10 April 1987, accepted 9 June 1987
The contractility of a helical strip of the thoracic aorta was studied in rats injected intraperitoneally with endotoxin. The contractile response to any of the agonistic agents, KC1, norepinephrine or 5-hydroxytryptamine was time dependently diminished in the endotoxin-injected rats compared to the controls. This diminution preceded the depression of blood pressure. When the external calcium concentration was increased from 2.5 to 7.5 mM after the KC1 (80 mM)-induced contractile response reached a plateau, the diminished contractile response was reversed in the endotoxin-injected group. The strips from the endotoxin-injected rats showed a higher 45CAC12uptake into the vascular tissue with the KCl-stimulated contraction. These findings suggest that the blood pressure depression during endotoxic shock may be attributed partially to the diminished contractility of the blood vessels and that this diminution is induced by a disorder of calcium utilization within vascular smooth muscle during vascular contraction. Endotoxin; Vasoconstriction; Calcium
1. Introduction Circulatory failure in sepsis exerts a serious effect on the prognosis and frequently does not respond to various types of therapeutic management. However, the precise mechanism of cardiovascular abnormality in endotoxic shock is still unknown. It has been reported recently that endotoxin treatment induces vascular hyporesponsiveness to catecholamines in vivo (Parratt, 1973; Auclair et al., 1982). On the other hand, in vitro evaluation of the vascular reactivity during endotoxic shock has so far been httle described. Pomerantz et al. (1982) reported that the contractile response to norepinephrine and prostaglandin endoperoxide was reduced in thoracic aorta removed from rats injected intravenously (i.v.) with endotoxin. The cause of this diminished contractile response in vitro is still unknown. Recent * To whom all correspondence should be addressed: Second Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-Cho, Nishinomiya, Hyogo, 663, Japan.
studies using cardiac preparations in vitro have demonstrated that endotoxin induces cardiac depression which is associated with alterations in calcium metabohsm in the heart (Hess et al., 1980; Parker and Adams, 1981). The purpose of this study was to investigate vasocontractility changes after endotoxin treatment. Thoracic aortas from rats were used for this purpose and also to examine the mechanism of the contractile changes associated with calcium metabolism. 2. Materials and methods 2.1, Administration of endotoxin
Male Wistar rats (350-450 g) under anesthesia with sodium pentobarbital (30 m g / k g ) were treated by intraperitoneal (i.p.) injection of 10 m g / k g of endotoxin (lipopolysaccharide B from E. coli 0 2 6 : B 6 Difco Laboratories) diluted in physiological saline. The controls were rats treated i.p. with the same volume of physiological saline.
0014-2999/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
118
A catheter was inserted into the left femoral artery of rats under anesthesia with i.p. sodium pentobarbital (30 mg/kg), and a pressure transducer (Stotham, Model 23Db) was connected to measure MAP.
tetraacetic acid). A scintillation mixture was added and the radioactivity was determined with a liquid scintillation counter (Packard Tri-Carb Model 460 Liquid scintillation spectrometer). The rate of Ca uptake was calculated as c.p.m./kg of the La-resistant 45Ca fraction divided by c.p.m./ /~mol Ca of the specific activity of 45Ca-containing medium.
2.3. Observation of isometric contraction ,
2.5. Substances
The rats were killed by a blow on the neck. The thoracic aorta was removed and immediately placed in Krebs-Ringer solution (mM: 118 NaC1, 4.7 KCI, 25 N a H C O 3, 1.2 KH2PO4, 1.2 MgSO 4 • 7H20, 2.5 CaC12 and 10 glucose; p H 7.4), and helical strip specimens of 2 × 15 mm were prepared; care was taken not to injure the intimal layer (Furchgott and Bhadrakom, 1953). Each specimen was carefully suspended between hooks in an organ chamber containing 10 ml of KrebsRinger solution with a gas stream (37 o C) of 95% 02-5% CO 2 and attached to a force transducer connected to a Nihon Kohden polygraph under a load of 1 g. After 1.5 h of stabilization, the isometric contraction was measured and expressed in terms of mg contractile force/rag wet tissue
The drugs used were: (-)-norepinephrine hydrochloride (Sigma Chemical), 5-hydroxytryptamine creatinine sulfate (Sigma Chemical) and 45CAC12 (New England Nuclear). The concentration of each drug was expressed as the final concentration in the bath.
2.2. Measurement of mean arterial blood pressure (MAP)
2.6. Statistics The results of the experiments are expressed as means + S.E.M. The maximum response (Emax) and ECs0, the concentration producing a halfmaximal response were determined graphically from individual dose-response curves. Statistical analyses were performed with Student's t-test and a P value less than 0.05 was taken as significant.
weight. 2.4. Measurement of tissue
45CaCl2 uptake
into vascular
A ring-shaped vascular strip 5 mm long was prepared from the aorta. After 1.5 h of stabilization in 5 ml of the above buffer not containing K H 2 P O 4 under a gas stream of 95% 02-5% CO2, the ratio of Ca uptake into the vascular strip was determined by means of the low temperature 3+ La method (Godfraind, 1976). The strip was treated with 0.4 /~Ci/ml of 45CAC12 together with the agonistic agent. After 5 min, the strip was taken out and immediately transferred into 2 ° C controlled 50 mM LaC13 solution (mM : 118 NaC1, 4.7 KC1, 1.2 MgC12, 10 glucose, 50 LaC13 and 15 Tris-maleate; pH 6.8). The wet weight of the strip was measured after a 60 min wash. The strip was then allowed to stand overnight in 2 ml of 20 mM E G T A (ethyleneglycol-bis-(2-aminoethylether)-
3. Results
3.1. Changes of mean arterial blood pressure after endotoxin administration Figure 1 shows the blood pressure changes before and after the i.p. injection of endotoxin. The blood pressure displayed no significant changes until 3 h after the endotoxin injection but fell to shock level at 6 h. Anesthesia with i.p. sodium pentobarbital had no effect on the blood pressure (data not shown). 3.2. Effect of endotoxin administration on aorta contractility Figure 2 shows the changes in the contractile responses induced by KC1, norepinephrine or 5hydroxytryptamine before and after the i.p. injec-
119 120
10mln$
B) Endotoxin
A) Control 100
o= t, 0.s
LL
8O E
o 60
40 KC~8OmM
20
,
,
0
15.
i 3
6
Time (hr)
Fig. 1. Time course of changes in mean arterial blood pressure (MAP) after intraperitoneal injection of 10 mg/kg endotoxin. Each point represents the mean_+ S.E. * Statistically significant (P < 0.05) differences compared to the values before endotoxin treatment, n = 8.
tion of endotoxin. The responses to each agonist diminished gradually after the endotoxin injection. The ECs0 values for each agonist with aortas from endotoxin-injected rats increased significantly from 1.5 h after injection of endotoxin (table 1). The involvement of calcium in the contractile response to KC1 was investigated next. The contractile response to 80 mM KC1 was markedly diminished in the endotoxin-injected group compared to the controls. However, an additional 5
KC~ 80raM
Fig. 3. Representative recordings of additional effect of 5 mM CaC12 on contractile response caused by 80 mM KCI. (A) Control, (B) 6 h after intraperitoneal injection of 10 m g / k g endotoxin. Vertical bars illustrate percentage enhancement of KC1 (80 mM)-induced contraction by addition of 5 mM CaCI 2. ([2) control, ([]) 6 h after endotoxin injection. * Statistically significant (P < 0.01) differences between responses in controls and endotoxin-injected animals.
mM CaC12 produced a marked enhancement of the contractility and the diminished contractile response to KC1 was reversed in the endotoxin-injected group (fig. 3).
3.3. Effect of endotoxin administration on calcium uptake of aorta The 5 min uptake of 45CAC12 into vascular tissue did not differ between the two groups during the static phase. However, upon stimulation with 80 mM KC1, the aorta strips from the endo-
A
C
!
B .........
~oo. °°S°°°°°°*°
.~
"
i ~
I~V~T
~. . . .
o--o ] h,s (n-s) olb-.-~ Sh,. In- s / ~ , , o . ~ , , ' *
~.°°
~...L--~
" ®
u.
...... KCJ~(raM)
/
..'.
...~ .............. 4
: ,::,, ,0-,.
,~-.
,;-.
,;-.
~orepim~nr,~ (M)
~'o-.
~0-.
5×1o'-;lo-',
s~lo-.
~-,
2xw"
5- Hydro~ytryptami~ (M)
Fig. 2. Effect of intraperitoneal injection of 10 m g / k g endotoxin on contractile responses upon exposure to cumulative additions of KCl (A), norepinephrine (B) or 5-hydroxytryptamine (C). Vertical lines indicate S.E. * Statistically significant (P < 0.05) differences between responses in controls and endotoxin-injected animals.
120 TABLE 1 EC50 values and maximum contractile effects of KCI, norepinephrine (NE) and 5-hydroxytryptamine (5-HT) in rat thoracic aorta before and after intraperitoneal injection of 10 m g / k g endotoxin. Values are means ___S.E. Drug
ECs0 (M) Before
1.5 h
3h
6h
KCI NE 5-HT
(2.34 :t: 0.23) × 10- 2 (6.215-0.24) x 10 -9 (1.96 + 0.16) × 10- s
(4.07 + 0.52) × 10- 2 . (2.08 +0.27) x 10 -8 * (3.49 5- 0.43) × 10 - s .
(5.51 + 0.54) × 10- 2 . (3.70+0.31)× 10 - s * (3.03 5- 0.31) × 10- s .
(5.86 + 0.48) × 10- 2 . (5.14+0.74) x 10 -8 * (4.05 5- 0.63) × 10 - s .
Drug
Maximum contraction (mg tension/mg wet tissue)
KC1 NE 5-HT
Before
1.5 h
3h
6h
166 _+10.6 215.1+12.6 202.4 5-12.8
139.5_+17.3 194.1_+18.5 175.4 + 10.3
82.65- 7.1 * 130.65- 9.8 * 97.8 _+14.2 *
80.35- 8.7 * 84.3-+11.4 * 79.3 __ 14.8 *
• Denotes significant difference between control and endotoxin-treated preparations (P < 0.05).
KC~
~,<~0.01 I l
p<0.0s I"-I
f
C~ control I ~ endotoxin
ba~l NS r~ 100
(A)
m
g~
0
(B)
Fig. 4. Effect of intraperitoneal injection of 10 m g / k g endotoxin on Ca uptake, 5 min after vehicle administration (basal) and 80 mM KC1 stimulation (KC1) (A), and isometric tension development, 5 rain after 80 mM KC1 stimulation (B). (0) control, ([]) 6 h after endotoxin injection. * P < 0.05, * * P < 0.01 compared to the values obtained before endotoxin treatment.
toxin-injected rats showed a higher 4~CaC12 uptake than the control tissues (fig. 4A). The 5 min contractile force after stimulation with 80 mM KCI was greatly diminished in the endotoxin-injected group (fig. 4B).
4. Discussion
The contractility of the aorta strips from endotoxin-treated rats was found to diminish gradually prior to the depression of blood pressure. This diminution of the contractile response can trigger circulatory failure in endotoxic shock. Seaman
and Greenway (1984) reported from an in vivo study on the contractility of the hepatic vein that the contractile response to nerve stimulation or infusion of norepinephrine or angiotensin II was diminished in the endotoxin-injected group. Auclair et al. (1986) suggested, on the basis of in vivo and in vitro studies, that endotoxin has a potent blocking action on vascular a-adrenoceptors. In the present in vitro study, the contractile response to norepinephrine and 5-hydroxytryptamine, which is thought to be composed of biphasic components, namely, a phasic component (mobilization of intracellular calcium) and a tonic component (influx of extracellular calcium due to opening of the calcium channel) (Godfraind and Kaba, 1972; Nakaki et al., 1985), was reduced and the KCl-induced contraction which is not mediated by receptor stimulation but depolarization of the cell membrane was also reduced in the endotoxin-injected group. These results suggest that the diminished vasocontractility caused by endotoxin treatment is due not only to abnormality of postsynaptic specific receptors but to alterations of the common contractile mechanism after stimulation of the receptors. Abnormality of the energy metabolism occurs during endotoxic shock, with a consequent inhibition of transmembranous ion migration (Schumer, 1984). The ATP-dependent calcium uptake in vascular smooth muscle, at the level of subcellular fractions, is damaged by the action of endotoxin
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(Soulsby et al., 1976; Soulsby et al., 1980). Pomeranz et al. (1982) reported that the contractile response to norepinephrine was reduced in thoracic aortas excised from rats treated by i.v. injection of endotoxin and they speculated that this diminution of the contractile response resuited from the decreased calcium uptake by microsomes and mitochondria, which are the main storage pool of calcium. In the present experiments, KCl-induced contraction, which is dependent on extracellular calcium (Godfraind and Kaba, 1972), was also diminished in the endotoxin-injected groups. Thus, reduction of the intracellular calcium reserve is probably not the reason for the diminution of the contractile response. When the external calcium concentration was increased after the KC1 (80 mM)-induced contractile response had reached a plateau, the diminished contractile response was reversed in the endotoxin-injected groups. Instead, in contrast to the marked diminution of the 5 rain contractile force after stimulation with 80 mM KC1 in the endotoxin-injected group, the rate of 45CaC12 uptake during corresponding period was enhanced in this group compared to the controls. The 43CaC12 uptake stimulated by KC1 corresponds to the calcium influx through the calcium channel due to depolarization of the plasma membrane (Godfraind and Kaba, 1972). Accordingly, the voltagedependent calcium channel might not be damaged in the endotoxin-injected group. Ives et al. (1986) recently reported that the hypotension in endotoxin-treated rats was reversed by a calcium channel agonist, BAY k 8644. These findings therefore suggest that, in the endotoxin-injected group, disturbance of intracellular calcium utilization occurs in association with the excitation-contraction coupling of the vascular smooth muscle. One possible explanation for the decreased intracellular calcium utilization is an enhanced sequestration of calcium from cytosol in endotoxemia rather than diminished sequestration. Recent studies have demonstrated that the vascular endothelium plays an obligatory role in the regulation of vascular tonus (Furchgott, 1983). The endothelium-dependent relaxing response to a c e t y l c h o l i n e ( 1 0 - 6 M ) , which was expressed as a
percentage depression of the contractile responses to norepinephrine (10 -7 M), did not differ between control and endotoxin-injected groups (control: 55.3 + 3.1%, 6 h after endotoxin injection: 56.3 + 3.3%), and the vasocontraction caused by KC1, norepinephrine or 5-hydroxytryptamine was not altered by removal of the endothelium in both groups (data not shown). Therefore, the decreased vasocontractility in endotoxin-injected rat aortas is not due to changes in endothelial modulation of vascular tonus. The present experiments led to the conclusions that, in endotoxin-injected rats, reduction of aortic contractility is caused by a disorder of calcium utilization in association with the intracellular excitation-contraction coupling of vascular smooth muscles and that diminution of the contractile response of vascular smooth muscle can trigger circulatory failure in endotoxic shock.
Acknowledgement The authors wish to thank Dr. Kazuo Nagai for his valuable advice.
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