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Interleukin-1β causes different levels of nitric oxide-mediated depression of contractility in different positions of rat thoracic aorta
Life Sciences, Vol. 64, No. 16, pp. 13~1381,1999 copylight 0 1959 Elscvier science Inc. Printed in the USA. All rights resewed CQM-32OS/W/$-see front matter
PI1 SOO24-3205(!W)OOO71-5
INTERLEUKIN-10 CAUSES DIFFERENT LEVELS OF NITRIC OXIDE-MEDIATED DEPRESSION OF CONTRACTILITY IN DIFFERENT POSITIONS OF RAT THORACIC AORTA Li Fang Lu and Ronald R. F&us* Department of Physiology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territory, Hong Kong (Received in final form January 11, 1999)
Summary Interleukin-1B (IL-l@ can be synthesized by macrophages, endothelial cells and vascular smooth muscle cells when stimulated by bacterial lipopolysaccharide (end.otoxin) during septic shock. The IL-18 levels in the blood vessel wall are also elevated in atherosclerosis. IL-H3 can cause induction of inducible nitric oxide synthase (WOS) expression in vascular smooth muscle cells and produce vasorelaxation, hypotension and ultimately tissue damage. We studied the depressions of vascular smooth muscle contractions at 3 hours after exposure to IL1B in different positions of rat thoracic aorta. The data show that the aortic rings from the cranial end of rat thoracic aorta had little response to IL-B (0.5 and 1.0 ng/ml) while those from the caudal end of thoracic aorta had larger depressant S-methylisothiourea sulfate (SMT), an iNOS inhibitor, completely response. blocked the depression of contraction caused by IL-ID in intact aortic rings. If the endothelium was removed from the aortic rings before exposure to IL-l& all rings from different parts of the thoracic aorta showed an equal amount of vasodepression. Thus, the difference in the depressant response of IL-H3 in d&rent portions of thoracic aorta is endothelium-dependent and involves induction of NOS.
Key Wordr interleukin-lp, nitric oxide, atherosclerosis, thoracic artery
Interleukin-ll3 (IL-l@ is one of the cytokines thought to play a role in causing disease. It is released from macrophages and some other cells like endothelial cells and vascular smooth muscle cells during septic shock (1,2). Patients with septic syndrome have elevated levels of interleukin-l(IL-1), interleukind @L-6), and tumor necrosis factor-a (TNFa) (3). Prolonged exposure of blood vessels to IL-1D decreases vascular tone and causes systemic vasodilation (1,4). .
CORRESPONDING AUTHOR: Ronald R. Fiscus, Department of Physiology, Faculty of Shatin, New Territory, Hong Kong. Medicine, The Chinese University of Hong Kong, Telephone: 852-2609-6780. Fax: 852-2603-5022. E-mail: [email protected].
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IL- 1, TNFa and interferon-y (IFN-y) are also actively produced and released from activated macrophages and T-lymphocytes during atherosclerosis and arterial injury following balloon angioplasty (5). Atherosclerosis is a chronically progressive process involving initiation of smooth muscle proliferation, migration and further intimal proliferation and deposition of matrix (6). Moyer et al. also showed that IL-la and D mRNAs were also expressed in foam cells, adherent leukocytes, vascular smooth muscle cells and endothelial cells in arteries during atherosclerosis (7).
Some of the effects of IL-1R on vascular smooth muscle is suggested to be mediated by nitric oxide (NO) (8,9). IL-1R activates the synthesis of inducible nitric oxide synthase (iNOS) in vascular smooth muscle cells. The overproduction of NO can cause prolonged vasorelaxation, severe hypotension and even tissue damage (10). The present study shows that IL-18 causes different levels of contractile depression in different positions of rat thoracic aorta and that this difference is endothelium-dependent and involves iNOS activity. Material and Methods Preparation of isolated aortic rings The treatment of the laboratory animals and the experimental protocols of the present study adhered to the guidelines of The Chinese University of Hong Kong and were approved by an Institutional Authority for Laboratory Animal Care. Healthy, male Sprague-Dawley rats (body weight = 240 - 280 g) were used. Heparin (1000 U/rat) was injected intraperitoneally 15 min before rats were decapitated and exsanguinated. Their thoracic aortae were removed and placed in modified Krebs-Ringer-Bicarbonate (KRB) solution containing 118.5 mM NaCl, 4.74 mM KCl, 1.18 mM MgS04, 1.18 mM K.I&PO.I, 2.5 mM CaC12, 24.9 mM NaHC03, 10 mM glucose, and 0.03 mM EDTA, and aerated with 95% 02 plus 5% CO2, as in previous experiments (11). After removing the adhering fat and connective tissue, the aortae were cut transversely into rings of 4 mm long. The first aortic ring was cut 2 mm away from the first branch near the aortic arch. Usually, four to six usable rings were obtained from one thoracic aorta. The endothelium was removed in some of the aortic rings by gently rubbing the intimal surface with stainless steel microforceps. The integrity or removal of the endothelium was checked by observing the vasorelaxant response to acetylcholine, described below. Measurement of contractile and relaxant responses in aortic rings The rings were suspended in organ baths containing 5 ml of KRB bubbled with 95% 02 plus 5% CO2 at 37°C using two stainless steel rods (0.275 mm diameter) and were set under a resting tension of 1 gram, as in previous experiments (11). The KRB was replaced with fresh Contractile and relaxant responses solution every 10 min during a 30 min equilibration period. were measured isometrically using force transducers (Grass, Model FT-03) and recorded on a physiological recorder (Grass Polygraph, Model 7). To test whether the endothelium was intact, each ring was stably contracted with phenylephrine (100 nM) and then exposed to acetylcholine (ACh, 100 nM). Only those rings with larger than 50% relaxant responses were considered to have intact endothelium. To check whether the endothelium was successfully removed by the rubbing procedure described above, each ring was contracted with 20 nM phenylephrine to a plateau and then 100 nM ACh was added. Only those rings that had no response to ACh were considered without functional endothelium.
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To lest the effects of IL@3 in aortic rings, we compared the difference in the dose-response relationship of phenylephrine in the aortic rings before and after incubation with IL-l& Before measuring the dose-response relationship of phenylephrine, the aortic rings were first exposed to 1 pM phenylephrine and washed out two times in order to obtain a stable response to phenylephrine in the following steps. The dose-response relationship of phenylephrine was first measured before exposure to IL-l& After washing out the phenylephrine and allowing the rings to return to baseline, the aortic rings were incubated with IL-B3 for one hour. Then IL-ID were washed out and the aortic rings were incubated with pure KIU3 solution for an additional two hours during which time the KRB solution was replaced every 20 min. The final dose-response determination was performed 3 hours after IL-18 was first administered. Contractions caused by phenylephrine were tested by increasing the concentration of phenylephrine in the organ chambers in cumulative half-log increments after a steady-state response was reached to each increment and was expressed as percentage of the maximum contraction to phenylephrine in the first measured dose-response curve. To 1:estthe involvement of iNOS, an iNOS inhibitor, S-methylisothiourea sulfate (SMT), was added before the beginning of the final dose-response determination. Chemicals and Drugs Rats; were supplied by a colony of Sprague-Dawley rats from the Laboratory Animal
Service Center, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China. Interleukinll3 (Human, Recombinant) and S-methylisothiourea sulfate were purchased L-phenylephrine from Calbiochem-Novabiochem Corporation (San Diego, CA, USA). (hydrochloride), acetylcholine (Chloride) and heparin (sodium salt) were purchased from Sigma Chemical Company. Statistical analysis The data were analyzed using one-way ANOVA and further analyzed using the student-
Newman-Keuls (S-N-K) test for multiple comparisons between treatment groups. A p value of ~0.05 was used to indicate significant difference between treatment group means. The data are presented as mean values f the standard error of the mean (SEM). The ‘n’ values given in the figure legends represent the number of individual rats used for providing the aortic rings in each treatment group. Results Different levels of contractile depression caused by IL-lf3 in d@erent positions aorta with intact endothelium
of rat thoracic
When the aortic rings were incubated with IL-H3 for one hour, followed by two hours incubation without IL-l& the contractility of the aortic rings have been reported to be depressed (1). However, in our experiments, we found that IL-1R caused different levels of depressant responses to IL-113in aortic rings from different positions of rat thoracic aorta when endothelium was intact (Fig. 1). We cut four aortic rings from the thoracic aorta, from the cranial end near the aortic arch to the caudal end near the abdomen. The frost, third and fourth rings were incubated with the same concentration of IL-1R (0.5 @ml) and the same time course, whereas the second ring was treated as a time control, incubated for the same time without IL- 1B. After the treatment, the LogECjO values of the phenylephrine-induced contractions of the four aortic rings were 7.33*0.11, 7.25~0.12, 6.72kO.08, 6.8OiO.09, respectively. The contractions of these four aortic rings to 1001nM phenylephrine were 58.25.8%, 57.8*3.3%, 25.5*5.1% and 31.2*6.7% (Panel B, Fig. 1). These numbers represent the percentage of the maximum contraction to phenylephrine in
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the first dose-response curve (before addition of IL-l@. The dose-response curve of the first aortic ring after IL-ll3 treatment was the same as that of the second ring, the time control. In contrast, the third and forth rings treated with the same dose of IL-10 had significantly depressed contractility. Thus, the ring nearest the aortic arch had a significantly smaller response to IL-10 than rings nearest the abdomen.
100
s
ring1
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ring2(con) ring3
L
; E S
50
-I
0
ring4
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IO-'
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Fig. 1 IL-l&induced depression of contraction in aortic rings (with endothelium) from different positions of rat thoracic aortic (n=9). Four aortic rings of 4 mm in length were cut from the cranial end, near the aortic arch, to the caudal end, near the abdomen and numbered as ringl, ring2, ring3 and ring4. The rings 1,3 and 4 were incubated with 0.5 @ml IL-1B for one hour followed by an additional two hours without IL-II& while ring 2 was treated as a time control not exposed to IL1l3. A. The dose response curves showing phenylephrine-induced contractions of the aortic rings after treatment. B. The contractility of aortic rings to 100 nM phenylephrine. *P
In another set of experiments, we also investigated the whole image of the responses to IL1s of rat aortic rings from the cranial end to the caudal end (Fig 2). The thoracic aorta was carefully cut into 6 segments which were 4 mm long and incubated with 1 ng/ml IL-18 for one hour, followed by two hours incubation in KRB solution without IL-M+. A clear trend of the decrease of contractility was shown in the dose-response curves to phenylephrine displayed by the aortic rings cut from the cranial to caudal end. Especially the first aortic ring showed significantly different contractility from the others following incubation with IL-18. This indicates that the depressant response to IL- ll3 is increasing along the length of the rat thoracic aorta from cranial to abdominal end.
Regional IL-p Response in Aorta
Vol. 64, No. 16,1999
n
l377
ring1
A ring2
Id-8
IO-6 Phenylephrine
v
ring3
l
ring4
0
ring5
q
ring6
10
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Fig. 2 A whole image of the IL-l&induced depression of contraction in aortic rings (with endothelium) from cranial to caudal ends of rat thoracic aorta (n=7). Rat thoracic aortae were cut into 6 segments, each of 4 mm in length, and sequentially labeled as rings 1 to 6. The aortic rings were incubated with 1 @ml IL-18 for 1 hr followed by two hours incubation without IL-l& A. The dose response curves of phenylephrine-induced contractions of the aortic rings after treatment. B. The contractility of aortic rings to 100 nM phenylephrine. *P
Vol. 64, No. 16,1999
Regional IL-p Response in Aorta
1378
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0
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104
.
.
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Phenylephrine (M) Fig. 3 The effect of SMT on the changes of contractility induced by IL- 1R in aortic rings (with endothelium) from different positions of rat thoracic aorta (n=6). Four aortic rings of 4 mm in length were cut from cranial end near aortic arch to the caudal end near abdomen and numbered as ringl, ring2, ring3 and ring4. The rings 1, 3 and 4 were incubated with 1 rig/ml IL-1B for one hour followed by an additional two hours without IL-113, while the ring 2 was treated as time control. SMT (100 uM) was added before measuring the dose response to phenylephrine after 3 hours incubation. The data represent the dose-response curve to phenylephrine of the aortic rings after treatment.
Discussion Our present study shows that IL-lb causes different levels of depression of contractile Removal of endothelium activity in different positions of rat thoracic aorta with endothelium. before exposure to IL-18 or incubation with the iNOS inhibitor (SMT) removed this difference in vasodepression in the different portions of aorta. IL-10 is believed to be one of the mediators in many cardiovascular diseases (3). There is evidence showing elevated production of IL-l during septic shock and atherosclerosis (3,5). IL-lb can produce a shock-like syndrome, characterized by hypotension and decreased vascular resistance, when administrated to animals (4). IL-l-treated rat aortic rings exhibit a diminished vascular contractile response to phenylephrine and potassium chloride (1). The IL-l-induced inhibition of contraction is believed to be mediated by induced synthesis of NO, one agent that is pathologically over-produced and causes hypotension during septic shock (9).
RegionalILp Responsein Aorta
Vol. 64, No. 16, 1999
1379
ring1 A ring2 v ring3 l ring4
n
IO"
IO”
IO-’
IO”
Phenylephrine (M) Fig. 4 Depression of contraction caused by IL-M in aortic rings (without endothelium) from different positions of rat thoracic aorta. Four aortic rings of 4 mm in length were cut from the cranial end, near the aortic arch, to the caudal end, near the abdomen, and numbered as ringl, ring2, ring3 and ring4. The aortic rings were removed of endothelium before they are mounted into the organ baths. Rings 1, 3 and 4 were incubated with 2 ng/ml IL-IS for one hour followed by an additional two hours without IL-IS, while ring 2 was treated as a time control. Note that the dose-response curves to phenylephrine are significantly shifted down and to the right in all aortic rings after IL-113treatment, as compared to the time control (ring 2). *P
growth factor (PDGF) and transforming growth factor-g). The later will favor the proliferation of
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smooth muscle cells (10). However, the over-production of NO by iNOS could cause harmful effects in diseases. Excess NO can be involved in a series of reactions producing deleterious metabolites or free radicals, like a-hydroxynitrosamines, N-nitrosamines, peroxynitrite (ONOO), ‘NO2 and ‘OH, which can damage tissue (10). From our experiments in rat thoracic aorta the area near the aortic arch has lower level of NO induction, which may have implication regarding vascular damage in diseases associated with IL-l&induced overproduction of NO. The regional difference of induction of NO production disappeared when endothelium was denuded. Therefore, some factors in the endothelium of the thoracic aorta near the aortic arch may inhibit the induction of iNOS or inhibit the relaxation effect of the induced NO. The role played by the endothelium in different areas of the aorta needs to be clarified. In summary, the results presented in this paper show differences in the vasodepression effect of IL-18 in different positions of rat thoracic aorta with endothelium. This difference in vasodepression effect appears to involve difference in induction of iNOS, because it is completely prevented by the iNOS inhibitor SMT. The endothelium appears to play a role in mediating this difference of depressant response caused by IL- 18. Thus, these data may give us some new insight into the pathology of cardiovascular diseases. Acknowledgment The author would like to thank the expert technical assistant provide by Mr. Alex W. K. Tu. This research project was supported by an RGC Earmarked Grant from the Research Grants Council of Hong Kong (No. CUHK 266/96M) awarded to R. R. F. Reference 1. D. BEASLEY, R.A. COHEN AND N.G. LEVMSKY, J. Clin. Invest. 83 331-335 (1989). 2. C.A. DINARELLO, FASEB J. 2 108-l 15 (1988). 3. L.C. CASEY, R.A. BALK AND R.C. BONE, Ann. Intern. Med. 119 771-778 (1993). 4. S. OKUSAWA, J.A. GELFAND, T. IKEJIMA, R.J. CONNOLLY AND C.A. DINARELLO, J. Clin. Invest. 81 1162-1172 (1988). 5. G.A. JOLY, V.B. SCHINI AND P.M. VANHOUTTE, J. Cardiovasc. Pharmacol. 20 (Suppl. 12) S151-S154 (1992). 6. P. LIBBY AND G.K. HANSSON, Lab. Invest. 64 5-15 (1991). 7. C.F. MOYER, D. SAJUTHI, H. TULLI AND J.K. WILLIAMS, Am. J. Pathol. 138 95 l-960 (1991). 8. J.F. FRENCH, L.E. LAMBERT AND D.C. DAGE, J. Pharmacol. Exp. Ther. 259 260-264 (1991). 9. D. BEASLEY, J.H. SCHWARTZ AND B.M. BRENNER, J. Clin. Invest. 87 602-608 (1991). 10. F.V. DEFEUDIS, Gen. Pharmacol. 26 667-680 (1995). 11. R.R. FISCUS, H.L. ZHOU, X. WANG, C. HAN, S. ALI, CD. JOYCE AND F. MURAD, Neuropeptides 20 (2) 133-143 (1991). 12. R.G. KILBOURN AND P. BELLONI, J. Nat. Cancer Inst. 82 772-776 (1990). 13. M.W. RADOMSKI, R.M.J. PALMER AND S. MONCADA, Proc. Natl. Acad. Sci. USA 87 10043-10047 (1990). 14. J.B. HIBBS, JR., Z. VAVRIN AND R.R. TAINTOR, J. Immunol. 138 550-565 (1987). 15. D.J. STUEHR AND M.A. MARLETTA, Cancer Res. 47 5590-5594 (1987). 16. A.G. NICOLSON, N.E. HAITES, N.G. MCKAY, H.M. WILSON, A.M. MACLEOD AND N. BENJAMIN, Biochem. Biophys. Res. Commun. 193 1269-1274 (1993).
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Regional IL-p Response in Aorta
17. R. BUSSE AND A. MOLSCH, FEBS Lett. 275 87-90 (1990). 18. S. MONCADA, Acta Physiol. Stand. 145 201-227 (1992).
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INTERLEUKIN-10 CAUSES DIFFERENT LEVELS OF NITRIC OXIDE-MEDIATED DEPRESSION OF CONTRACTILITY IN DIFFERENT POSITIONS OF RAT THORACIC AORTA Li Fang Lu and Ronald R. F&us* Department of Physiology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territory, Hong Kong (Received in final form January 11, 1999)
Summary Interleukin-1B (IL-l@ can be synthesized by macrophages, endothelial cells and vascular smooth muscle cells when stimulated by bacterial lipopolysaccharide (end.otoxin) during septic shock. The IL-18 levels in the blood vessel wall are also elevated in atherosclerosis. IL-H3 can cause induction of inducible nitric oxide synthase (WOS) expression in vascular smooth muscle cells and produce vasorelaxation, hypotension and ultimately tissue damage. We studied the depressions of vascular smooth muscle contractions at 3 hours after exposure to IL1B in different positions of rat thoracic aorta. The data show that the aortic rings from the cranial end of rat thoracic aorta had little response to IL-B (0.5 and 1.0 ng/ml) while those from the caudal end of thoracic aorta had larger depressant S-methylisothiourea sulfate (SMT), an iNOS inhibitor, completely response. blocked the depression of contraction caused by IL-ID in intact aortic rings. If the endothelium was removed from the aortic rings before exposure to IL-l& all rings from different parts of the thoracic aorta showed an equal amount of vasodepression. Thus, the difference in the depressant response of IL-H3 in d&rent portions of thoracic aorta is endothelium-dependent and involves induction of NOS.
Key Wordr interleukin-lp, nitric oxide, atherosclerosis, thoracic artery
Interleukin-ll3 (IL-l@ is one of the cytokines thought to play a role in causing disease. It is released from macrophages and some other cells like endothelial cells and vascular smooth muscle cells during septic shock (1,2). Patients with septic syndrome have elevated levels of interleukin-l(IL-1), interleukind @L-6), and tumor necrosis factor-a (TNFa) (3). Prolonged exposure of blood vessels to IL-1D decreases vascular tone and causes systemic vasodilation (1,4). .
CORRESPONDING AUTHOR: Ronald R. Fiscus, Department of Physiology, Faculty of Shatin, New Territory, Hong Kong. Medicine, The Chinese University of Hong Kong, Telephone: 852-2609-6780. Fax: 852-2603-5022. E-mail: [email protected].
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IL- 1, TNFa and interferon-y (IFN-y) are also actively produced and released from activated macrophages and T-lymphocytes during atherosclerosis and arterial injury following balloon angioplasty (5). Atherosclerosis is a chronically progressive process involving initiation of smooth muscle proliferation, migration and further intimal proliferation and deposition of matrix (6). Moyer et al. also showed that IL-la and D mRNAs were also expressed in foam cells, adherent leukocytes, vascular smooth muscle cells and endothelial cells in arteries during atherosclerosis (7).
Some of the effects of IL-1R on vascular smooth muscle is suggested to be mediated by nitric oxide (NO) (8,9). IL-1R activates the synthesis of inducible nitric oxide synthase (iNOS) in vascular smooth muscle cells. The overproduction of NO can cause prolonged vasorelaxation, severe hypotension and even tissue damage (10). The present study shows that IL-18 causes different levels of contractile depression in different positions of rat thoracic aorta and that this difference is endothelium-dependent and involves iNOS activity. Material and Methods Preparation of isolated aortic rings The treatment of the laboratory animals and the experimental protocols of the present study adhered to the guidelines of The Chinese University of Hong Kong and were approved by an Institutional Authority for Laboratory Animal Care. Healthy, male Sprague-Dawley rats (body weight = 240 - 280 g) were used. Heparin (1000 U/rat) was injected intraperitoneally 15 min before rats were decapitated and exsanguinated. Their thoracic aortae were removed and placed in modified Krebs-Ringer-Bicarbonate (KRB) solution containing 118.5 mM NaCl, 4.74 mM KCl, 1.18 mM MgS04, 1.18 mM K.I&PO.I, 2.5 mM CaC12, 24.9 mM NaHC03, 10 mM glucose, and 0.03 mM EDTA, and aerated with 95% 02 plus 5% CO2, as in previous experiments (11). After removing the adhering fat and connective tissue, the aortae were cut transversely into rings of 4 mm long. The first aortic ring was cut 2 mm away from the first branch near the aortic arch. Usually, four to six usable rings were obtained from one thoracic aorta. The endothelium was removed in some of the aortic rings by gently rubbing the intimal surface with stainless steel microforceps. The integrity or removal of the endothelium was checked by observing the vasorelaxant response to acetylcholine, described below. Measurement of contractile and relaxant responses in aortic rings The rings were suspended in organ baths containing 5 ml of KRB bubbled with 95% 02 plus 5% CO2 at 37°C using two stainless steel rods (0.275 mm diameter) and were set under a resting tension of 1 gram, as in previous experiments (11). The KRB was replaced with fresh Contractile and relaxant responses solution every 10 min during a 30 min equilibration period. were measured isometrically using force transducers (Grass, Model FT-03) and recorded on a physiological recorder (Grass Polygraph, Model 7). To test whether the endothelium was intact, each ring was stably contracted with phenylephrine (100 nM) and then exposed to acetylcholine (ACh, 100 nM). Only those rings with larger than 50% relaxant responses were considered to have intact endothelium. To check whether the endothelium was successfully removed by the rubbing procedure described above, each ring was contracted with 20 nM phenylephrine to a plateau and then 100 nM ACh was added. Only those rings that had no response to ACh were considered without functional endothelium.
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To lest the effects of IL@3 in aortic rings, we compared the difference in the dose-response relationship of phenylephrine in the aortic rings before and after incubation with IL-l& Before measuring the dose-response relationship of phenylephrine, the aortic rings were first exposed to 1 pM phenylephrine and washed out two times in order to obtain a stable response to phenylephrine in the following steps. The dose-response relationship of phenylephrine was first measured before exposure to IL-l& After washing out the phenylephrine and allowing the rings to return to baseline, the aortic rings were incubated with IL-B3 for one hour. Then IL-ID were washed out and the aortic rings were incubated with pure KIU3 solution for an additional two hours during which time the KRB solution was replaced every 20 min. The final dose-response determination was performed 3 hours after IL-18 was first administered. Contractions caused by phenylephrine were tested by increasing the concentration of phenylephrine in the organ chambers in cumulative half-log increments after a steady-state response was reached to each increment and was expressed as percentage of the maximum contraction to phenylephrine in the first measured dose-response curve. To 1:estthe involvement of iNOS, an iNOS inhibitor, S-methylisothiourea sulfate (SMT), was added before the beginning of the final dose-response determination. Chemicals and Drugs Rats; were supplied by a colony of Sprague-Dawley rats from the Laboratory Animal
Service Center, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China. Interleukinll3 (Human, Recombinant) and S-methylisothiourea sulfate were purchased L-phenylephrine from Calbiochem-Novabiochem Corporation (San Diego, CA, USA). (hydrochloride), acetylcholine (Chloride) and heparin (sodium salt) were purchased from Sigma Chemical Company. Statistical analysis The data were analyzed using one-way ANOVA and further analyzed using the student-
Newman-Keuls (S-N-K) test for multiple comparisons between treatment groups. A p value of ~0.05 was used to indicate significant difference between treatment group means. The data are presented as mean values f the standard error of the mean (SEM). The ‘n’ values given in the figure legends represent the number of individual rats used for providing the aortic rings in each treatment group. Results Different levels of contractile depression caused by IL-lf3 in d@erent positions aorta with intact endothelium
of rat thoracic
When the aortic rings were incubated with IL-H3 for one hour, followed by two hours incubation without IL-l& the contractility of the aortic rings have been reported to be depressed (1). However, in our experiments, we found that IL-1R caused different levels of depressant responses to IL-113in aortic rings from different positions of rat thoracic aorta when endothelium was intact (Fig. 1). We cut four aortic rings from the thoracic aorta, from the cranial end near the aortic arch to the caudal end near the abdomen. The frost, third and fourth rings were incubated with the same concentration of IL-1R (0.5 @ml) and the same time course, whereas the second ring was treated as a time control, incubated for the same time without IL- 1B. After the treatment, the LogECjO values of the phenylephrine-induced contractions of the four aortic rings were 7.33*0.11, 7.25~0.12, 6.72kO.08, 6.8OiO.09, respectively. The contractions of these four aortic rings to 1001nM phenylephrine were 58.25.8%, 57.8*3.3%, 25.5*5.1% and 31.2*6.7% (Panel B, Fig. 1). These numbers represent the percentage of the maximum contraction to phenylephrine in
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the first dose-response curve (before addition of IL-l@. The dose-response curve of the first aortic ring after IL-ll3 treatment was the same as that of the second ring, the time control. In contrast, the third and forth rings treated with the same dose of IL-10 had significantly depressed contractility. Thus, the ring nearest the aortic arch had a significantly smaller response to IL-10 than rings nearest the abdomen.
100
s
ring1
E ._
ring2(con) ring3
L
; E S
50
-I
0
ring4
Id-6
IO-'
10-6
I&”
Phenylephrine (M)
Fig. 1 IL-l&induced depression of contraction in aortic rings (with endothelium) from different positions of rat thoracic aortic (n=9). Four aortic rings of 4 mm in length were cut from the cranial end, near the aortic arch, to the caudal end, near the abdomen and numbered as ringl, ring2, ring3 and ring4. The rings 1,3 and 4 were incubated with 0.5 @ml IL-1B for one hour followed by an additional two hours without IL-II& while ring 2 was treated as a time control not exposed to IL1l3. A. The dose response curves showing phenylephrine-induced contractions of the aortic rings after treatment. B. The contractility of aortic rings to 100 nM phenylephrine. *P
In another set of experiments, we also investigated the whole image of the responses to IL1s of rat aortic rings from the cranial end to the caudal end (Fig 2). The thoracic aorta was carefully cut into 6 segments which were 4 mm long and incubated with 1 ng/ml IL-18 for one hour, followed by two hours incubation in KRB solution without IL-M+. A clear trend of the decrease of contractility was shown in the dose-response curves to phenylephrine displayed by the aortic rings cut from the cranial to caudal end. Especially the first aortic ring showed significantly different contractility from the others following incubation with IL-18. This indicates that the depressant response to IL- ll3 is increasing along the length of the rat thoracic aorta from cranial to abdominal end.
Regional IL-p Response in Aorta
Vol. 64, No. 16,1999
n
l377
ring1
A ring2
Id-8
IO-6 Phenylephrine
v
ring3
l
ring4
0
ring5
q
ring6
10
(M)
Fig. 2 A whole image of the IL-l&induced depression of contraction in aortic rings (with endothelium) from cranial to caudal ends of rat thoracic aorta (n=7). Rat thoracic aortae were cut into 6 segments, each of 4 mm in length, and sequentially labeled as rings 1 to 6. The aortic rings were incubated with 1 @ml IL-18 for 1 hr followed by two hours incubation without IL-l& A. The dose response curves of phenylephrine-induced contractions of the aortic rings after treatment. B. The contractility of aortic rings to 100 nM phenylephrine. *P
Vol. 64, No. 16,1999
Regional IL-p Response in Aorta
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H ring1 A ring2(con) v ring3 l ring4
0
‘.
a,.
104
.
.
.11111,
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,,,,vr,
.
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,
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106
Phenylephrine (M) Fig. 3 The effect of SMT on the changes of contractility induced by IL- 1R in aortic rings (with endothelium) from different positions of rat thoracic aorta (n=6). Four aortic rings of 4 mm in length were cut from cranial end near aortic arch to the caudal end near abdomen and numbered as ringl, ring2, ring3 and ring4. The rings 1, 3 and 4 were incubated with 1 rig/ml IL-1B for one hour followed by an additional two hours without IL-113, while the ring 2 was treated as time control. SMT (100 uM) was added before measuring the dose response to phenylephrine after 3 hours incubation. The data represent the dose-response curve to phenylephrine of the aortic rings after treatment.
Discussion Our present study shows that IL-lb causes different levels of depression of contractile Removal of endothelium activity in different positions of rat thoracic aorta with endothelium. before exposure to IL-18 or incubation with the iNOS inhibitor (SMT) removed this difference in vasodepression in the different portions of aorta. IL-10 is believed to be one of the mediators in many cardiovascular diseases (3). There is evidence showing elevated production of IL-l during septic shock and atherosclerosis (3,5). IL-lb can produce a shock-like syndrome, characterized by hypotension and decreased vascular resistance, when administrated to animals (4). IL-l-treated rat aortic rings exhibit a diminished vascular contractile response to phenylephrine and potassium chloride (1). The IL-l-induced inhibition of contraction is believed to be mediated by induced synthesis of NO, one agent that is pathologically over-produced and causes hypotension during septic shock (9).
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ring1 A ring2 v ring3 l ring4
n
IO"
IO”
IO-’
IO”
Phenylephrine (M) Fig. 4 Depression of contraction caused by IL-M in aortic rings (without endothelium) from different positions of rat thoracic aorta. Four aortic rings of 4 mm in length were cut from the cranial end, near the aortic arch, to the caudal end, near the abdomen, and numbered as ringl, ring2, ring3 and ring4. The aortic rings were removed of endothelium before they are mounted into the organ baths. Rings 1, 3 and 4 were incubated with 2 ng/ml IL-IS for one hour followed by an additional two hours without IL-IS, while ring 2 was treated as a time control. Note that the dose-response curves to phenylephrine are significantly shifted down and to the right in all aortic rings after IL-113treatment, as compared to the time control (ring 2). *P
growth factor (PDGF) and transforming growth factor-g). The later will favor the proliferation of
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smooth muscle cells (10). However, the over-production of NO by iNOS could cause harmful effects in diseases. Excess NO can be involved in a series of reactions producing deleterious metabolites or free radicals, like a-hydroxynitrosamines, N-nitrosamines, peroxynitrite (ONOO), ‘NO2 and ‘OH, which can damage tissue (10). From our experiments in rat thoracic aorta the area near the aortic arch has lower level of NO induction, which may have implication regarding vascular damage in diseases associated with IL-l&induced overproduction of NO. The regional difference of induction of NO production disappeared when endothelium was denuded. Therefore, some factors in the endothelium of the thoracic aorta near the aortic arch may inhibit the induction of iNOS or inhibit the relaxation effect of the induced NO. The role played by the endothelium in different areas of the aorta needs to be clarified. In summary, the results presented in this paper show differences in the vasodepression effect of IL-18 in different positions of rat thoracic aorta with endothelium. This difference in vasodepression effect appears to involve difference in induction of iNOS, because it is completely prevented by the iNOS inhibitor SMT. The endothelium appears to play a role in mediating this difference of depressant response caused by IL- 18. Thus, these data may give us some new insight into the pathology of cardiovascular diseases. Acknowledgment The author would like to thank the expert technical assistant provide by Mr. Alex W. K. Tu. This research project was supported by an RGC Earmarked Grant from the Research Grants Council of Hong Kong (No. CUHK 266/96M) awarded to R. R. F. Reference 1. D. BEASLEY, R.A. COHEN AND N.G. LEVMSKY, J. Clin. Invest. 83 331-335 (1989). 2. C.A. DINARELLO, FASEB J. 2 108-l 15 (1988). 3. L.C. CASEY, R.A. BALK AND R.C. BONE, Ann. Intern. Med. 119 771-778 (1993). 4. S. OKUSAWA, J.A. GELFAND, T. IKEJIMA, R.J. CONNOLLY AND C.A. DINARELLO, J. Clin. Invest. 81 1162-1172 (1988). 5. G.A. JOLY, V.B. SCHINI AND P.M. VANHOUTTE, J. Cardiovasc. Pharmacol. 20 (Suppl. 12) S151-S154 (1992). 6. P. LIBBY AND G.K. HANSSON, Lab. Invest. 64 5-15 (1991). 7. C.F. MOYER, D. SAJUTHI, H. TULLI AND J.K. WILLIAMS, Am. J. Pathol. 138 95 l-960 (1991). 8. J.F. FRENCH, L.E. LAMBERT AND D.C. DAGE, J. Pharmacol. Exp. Ther. 259 260-264 (1991). 9. D. BEASLEY, J.H. SCHWARTZ AND B.M. BRENNER, J. Clin. Invest. 87 602-608 (1991). 10. F.V. DEFEUDIS, Gen. Pharmacol. 26 667-680 (1995). 11. R.R. FISCUS, H.L. ZHOU, X. WANG, C. HAN, S. ALI, CD. JOYCE AND F. MURAD, Neuropeptides 20 (2) 133-143 (1991). 12. R.G. KILBOURN AND P. BELLONI, J. Nat. Cancer Inst. 82 772-776 (1990). 13. M.W. RADOMSKI, R.M.J. PALMER AND S. MONCADA, Proc. Natl. Acad. Sci. USA 87 10043-10047 (1990). 14. J.B. HIBBS, JR., Z. VAVRIN AND R.R. TAINTOR, J. Immunol. 138 550-565 (1987). 15. D.J. STUEHR AND M.A. MARLETTA, Cancer Res. 47 5590-5594 (1987). 16. A.G. NICOLSON, N.E. HAITES, N.G. MCKAY, H.M. WILSON, A.M. MACLEOD AND N. BENJAMIN, Biochem. Biophys. Res. Commun. 193 1269-1274 (1993).
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17. R. BUSSE AND A. MOLSCH, FEBS Lett. 275 87-90 (1990). 18. S. MONCADA, Acta Physiol. Stand. 145 201-227 (1992).
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