Abstracts
T. F. Wischer, Basel T. Masaki, Kyoto M .J. Mulvany, Aarhus G .M . Ruhanyi, Berlin J . R. Varie, London P.M . Vanhoutte, Houston. Tex.
2nd International Symposium on Endothelium-Derived Vasoactive Factors
Editors
Basel, Switzerland, April 22-25,1992
1
2
Contractions to Endothelin-1 in Canine Coronary Arteries Are Inhibited by Sympathetic Activation
Vasa vasorum and Endothelial Cell Function in Veno-Venous Grafts
P. Aarnio, C.G.A. McGregor, V.M. Miller
G. Adamczyk, K. Lemhowicz, M. Walski, W.L. Olszewski
Department o f Surgery and Department o f Physiology and Biophysics. Mayo Clinic and Foundation. Rochester. M inn.. U SA
Surgical Research and Transplantation Laboratory, Experimental and Clinical Medical Research Centre. Polish Academy o f Sciences, and Scanning Electron Microscopy U nit. The Centre o f Child Health, Warsaw. Poland
The endothelium-derived factor endothelin-1 contracts vascular smooth muscle and inhibits release o f neurotransmitter from adren ergic and cholinergic neurons. The interaction o f these mechanisms in a blood vessel which receives both adrenergic and cholinergic innervation was studied. Canine left anterior descending coronary arteries were cut into rings, the endothelium was removed and the rings were suspended for the measurement o f isometric force in organ chambers. Endothelin-1 caused concentration-dependent con tractions in all rings. During electrical stimulation ( I Hz. 9 V . 2 ms) to activate sympathetic and parasympathetic nerve endings, the con tractions to endothelin-1 were reduced significantly. This decrease was greater in the presence o f atropine (1 X 10 6 M) and was elimi nated by a combination o f phentolamine ( 1 X 1 O' 6 M ) and propran olol ( 5 X I O *6M ). These results suggest that contractions to endothe lin-1 are reduced by transmitter released from sympathetic nerve endings. To determine the effect o f endothelin-1 presynaptically, superperfusion studies were performed. Electrical stimulation signif icantly increased release o f norepinephrine. Endothelin-1 inhibited the release o f tritium by electrical stimulation only at high concentra tions o f endothelin-1 (4 X 10"7 M). both in the absence and presence o f atropine. These results suggest that in canine coronary arteries presynaptic effects o f endothelin -1 are minimal and affect sympathetic neurotransmission. Together, the results suggest that contractions to endothelin -1 are modulated by sympathetic and parasympathetic tone postsynaptically. If these results can be generalized to human coronary arteries, then endothelin -1 may have more profound effects on vascular resistance in denervated hearts, for example, those used for transplantation.
Vasa vasorum (VV) and other structures o f the vascular wall arc damaged during harvesting the vein for grafting. This process leads to partial detachment o f endothelium (EC) and impairment o f its function. Aim o f the study was to investigate the relationship between the revascularisation o fV V o f the venous autograft and the recovery o f E C functions measured in a prostacyclin (P G L ) and fibrinolytic activity assay. The study was carried out on mongrel dogs o f both sexes weighing ==? 18 kg. The technique o f graft harvesting described by LoGcrfo was used. The study was carried out in 6 exper imental groups: ( 1 ) venovenous grafts. (2) venous grafts wrapped with plastic foil to hinder the regrowth o f V V , (3) venous grafts ligated. (4) in situ veins dissected from surrounding tissues. (5) in situ veins wrapped with plastic foil, and (6) in situ veins occluded by ligation o f both ends. Blood flow through V V was measured with use o f albumin microspheres labelled with 99mTc. Fibrinolytic activity was measured on standard fibrin plates with modified Todds tech nique. P G L production was measured in a RIA for P G F |„. Microan giograms were performed with use o f diaminobenzidine staining for endogenous peroxidase o f red cells. Scanning electron microscopy was performed. Results were expressed in percentages o f control values obtained from a nonoperated vein o f the same animal. Blood flow through VV in group 1 was 19% on day 1. 26% on day 5 and 67% on day 7. In group 2 it was 8 % on day 1.8 % on day 5, 51 % on day 7; i n group 3.3, 76 and 181 %; in group 4 30, 9 and 141 %; in group 5 21.5 and 292% and in group 6 29, 238 and 321 %. respectively. Fibrinolytic activity was in group 1 595% onday 1 .4 2 % o n d ay4 to 126% on day 7: it was
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J Vase Rcs 1992:29:75-232
in group 2 287, 110 and 86%; in group 3 20. 70 and 36%: in group 4 50, 231 and 155%; in group 5 34. 104 and 375%; in group 6 23. 33. and 57%, respectively. P G I 2 activity was in group 1 180% on day 1. 30% on day 4. and 123% on day 7; in group 2 it was 436, 282 and 87%; in group 3 241, 57% and 5%: in group 4 86, 137 and 43%; in group 5 320, 283 and 78%, respectively, and in group 6 1153% on day 1.2 2 6 % on day 4 and 30% on day 7. Microangiography revealed the ingrowth o f V V in group 1, on day 6. and the beginning o f their differentiation into venules and arterioles on day 7. Scanning elec tron microscopy presented full endothelial coverage in group 1 .4 , 5 and 6. The number o f E C in group I was 86% on day 6. Conclusions: Revascularisation o f V V and llow through the lumen o f venovenous grafts are necessary for maintenance o f endo thelial cell functions. Neither restoration o f flow through V V nor lumen only are satisfactory.
acetylcholine produces pulmonary vasodilation and alters V A/Q rela tionships in patients with chronic obstructive lung disease. Breathing N O induces marked and selective pulmonary vasodilation with no changes in systemic hemodynamics and no alterations in arterial oxygenation.
4
Endothelin-Converting Enzyme from Bovine and Human Endothelial Cells Kyunghye Ahn, Karen Beningo, Gregory Olds. Donald Hope Department o f Biochemistry. Pharmaceutical Research Division. Parke-Davis/Warner-Lambert Company. Ann Arbor. M ich.. U SA
Responses to Infusion of Acetylcholine and Inhalation of Nitric Oxide in Patients w ith Chronic Lung Disease and Pulmonary Hypertension 5. Adnot, C. Kouyoumdjian, M.D. Fratacci. C. Defouilloy,
P. Andrivet, S. Sediame, R. Herigaut Departement de Physiologie, IN SE R M U296, Hôpital H . Mondor, Créteil, France Endothelium-dependent relaxation in the pulmonary circulation is abolished in experimental animals during chronic exposure to hyp oxia. Moreover, isolated large pulmonary arteries o f patients with chronic lung disease show impaired relaxation to acetylcholine. We examined the responses to incremental infusion rates o f acetylcho line (5, 10 and 15 mg/min) and to inhalation o f nitric oxide (NO) at the increasing concentrations o f 5. 10. 20. and 40 ppm on hemody namics and gas exchange in 6 patients with severe chronic obstruc tive lung disease (5 m, 1 f, mean age: 56 ± 4 years). Infusion o f ace tylcholine at 15 mg/min increased cardiac index from 3.8 ± 0.4 to 4.6 ± 0.4 l/min/m2 (p < 0.01) with no change in pulmonary' artery pressure (Pap) (27 ± 1 vs. 28 ± 1 mm Hg. N S), systemic arterial pressure (Sap) ( 102 ± 3 vs. 105 ± 2 mm Hg, NS), heart rate (HR) (94 ± 5 vs. 93 ± 5 p/min, NS), cardiac filling pressures and ventilation. Pulmonary' and systemic vascular resistance decreased in a dosedependent manner, from 4.7 ± 0.4 to 3.6 ± 0.3 IU (p < 0.01 ) and from 26.5 ± 2.5 to 21 ± 5 IU (p < 0.01). respectively. Acetylcholine reduced P a 0 2 (from 56 ± 2 to 50 ± 2 mm Hg. p < 0.01 ), S a 0 2(from 85.5 ± 2.5 to 80.5 ± 2.5%, p < 0.01), S v 0 2 (from 66 ± 2 to 63 ± 2%. p < 0.05). arteriovenous 0 2 difference (from 4.1 ± 0.3 to 3.7 ± 0.3%, p < 0.05), and increased venous admixture (Q va/Q t ) (from 34.7 ± 3 to 45.7 ± 3%. p < 0.01). In contrast, breathing N O at 40 ppm decreased Pap from 29 ± 1 to 24 ± 1 mm Hg (p < 0.01 ). P V R from 4.2 ± 0.3 to 3.0 ± 0.2 IU (p < 0.01) with no other associated hemodynamic changes. Arterial P 0 2 remained unchanged or even tended to increase (from 59 ± 3 to 62 ± 3 mm Hg, NS), with a marked increase in some individual patients; Q va/Q i tended to decrease, from 34.6 ± 3.3 to 30.6 ± 3.0% (NS). We concluded that
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5
Potentiation of the Antiaggregatory Action of Organic Nitrates by Endothelial and Smooth Muscle Cells Is Due to Formation of Nitric Oxide ./. Aissa, M. Feelisclt Department o f Pharmacology. Schwarz Pharma. Monheim. Germany Organic nitrates exert profound vasodilator effects in vitro as well as in vivo. Former attempts to convincingly demonstrate additional antiplatelet activity in vitro, however, failed. Since cultured vascular cells have recently been demonstrated to effectively metabolize organic nitrates to N O and since N O is known to inhibit platelet aggregation, we reinvestigated the antiplatelct potential o f nitrates in
2nd Int Svmpon Endothelium-Derived Vasoactive Factors
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3
Endothelin-1 (ET-1). a highly potent vasocontractive 21-residue peptide, is formed following specific cleavage between Trp 21 and V al 22 o f the intermediate peptide ‘big E T -F (1-39 for porcine and 1-38 for human) by the putative endothclin-converting enzyme (ECE). We have identified and characterized an E C E activity from bovine and human endothelial cells. Our data clearly demonstrate that E C E is a membrane-bound metalloproteasc: substantial inhibi tion (96-98%) by metalloproteasc inhibitors (E D T A , phosphoramidon. and phenanthroline) was observed, whereas little or no inhibi tion was found for pepstatin A, leupeptin. P M SF . soybean trypsin inhibitor, or E-64. The bovine enzyme, which can be solubilized in Triton X-100. has a pH optimum o f 7.2 and a K m o f 20 \iM for big E T -1. The reaction was linear for at least 5 h. The E C E activity eluted at a volume which would correspond to a M W o f 400 kDa by gel filtration. We have elucidated the binding region o f big E T -1 for ECE and will report our progress toward the purification o f ECE. Thus. E CE is a membrane-bound metalloprotease with a putative molecular weight o f 400 kDa. Purification o f the enzyme may allow to develop specific inhibitors.
6
Release of Human Umbilical Endothelium-Derived Relaxing Factor in Pregnancy-Induced Hypertension F. Akar3. M. Ark3, B.S. Hydes3. M.E. Soysalb. F SaraçoghC. N. Abacioglu3. J. van de Voordec. / Kanzik3 3Dept. Pharmacology, Fac. Pharmacy G azi U n iv ., and bAnkara Maternity Hospital, Ankara, Turkey; cLab. Normal and Path. Physiol., U niv. Ghent. Belgium The physiological abnormalities o f preeclampsia can be ex plained by dysfunction o f vascular endothelium. The present investi gation was designed to determine whether umbilical E D R F biosyn thesis differs in the normotensive pregnant and preeclamptic wom en. E D R F release from umbilical vessels (U V . donor tissues) in both groups were compared by using a cascade bioassay system. The rat aortic rings (R A R . detector tissue) were precontracted with nor adrenaline (10 ~7 M) and the EDRF-induccd relaxation in response
to histamine (HA) or bradykinin (BK) was expressed as a percentage o f precontracted tone. Addition o f H A (10 -8 —1 0 ° M ) or BK (10 -9 — 1O' 6M) to the perfusion system o f U V caused dose-dependent relax ations o f the precontracted R A R . The relaxations were inhibited with nitroarginine (I0 ~4 M) but not with indomethacin (5 X 10~5 M). In the preeclamptic group. BK-induced E D R F release was reduced significantly as compared with that o f normotensive group (57.4 ± 8.1 vs. 22.4 ± 4.2%. n = 4) in the artery and (50.4 ± 12.3 vs. 16.2 ± 5.9%, n = 4) in the vein. On the other hand. HA-induced E D R F release did not change when compared with the normotensive group (59.1 ± 8.3 vs. 39.2 ± 6.1 %. n = 5) in the artery and (56.3 ± 12.3 vs. 39.6 ± 8.2%. n = 5) in the vein. Our findings indicate a probable change in endothelial cell (EC) function but not in E C integ rity in precclampsia. Supported by N A T O grant No. CRG-900104.
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Comparison of Endothelium-Dependent Responses to Histamine of Human Gastroepiploic Artery and Saphenous Vein /•'. Akar3. B.S. Uydes3. K. AxranaoghC. A. Yenerb, S. Aflainacic. M. Arsond, A. Toriiner**. ¡Kanzik3 3Dept. Pharmacology, Fac. Pharmacy, G azi U n iv ., bDept. Cardiovascular Surgery. Fac. Medicine, G azi U n iv ., 'Turkish Organ Transplantation and Bum Foundation Hospital, and J Dept. General Surgery, Fac. Medicine, Ankara U n iv ., Ankara, Turkey The use o f gastroepiploic artery (GEA) for coronary artery' bypass grafting has been described recently. The present study was designed to compare the endothelium-dependent responses o f G E A and saphenous vein (SV) to histamine. SV and G E A were supplied by patients undergoing operation. G E A and SV rings were precon tracted with noradrenaline. SV relaxed slightly (5.3 ± 1.9%) to lower doses o f histamine ( 10 '8- 10"7 M), which disappeared with nitroar ginine (N O A R G . K H M) or denudation while it contracted to higher doses (10 -6 - 10-4 M). On the other hand, in G E A histamine ( 1O' 9- 1O' 4 M ) caused only relaxations which occurred dose-dependently (91.5 ± 6.3%). These relaxations were inhibited partially with either N O A R G . indomethacin (5 X 10“6 M) and cimetidine (10~4 M) or denudation. Histamine also caused relaxation in G E A even in the presence o f the combination o f all above antagonists (32.8 ± 10.7%). Complete inhibition was only observed with the combina tion o f the histamine receptor antagonists cimetidine and mepyramine ( 10 -5 M). These results show that in G E A histamine causes a completely different response profile than that o f SV .
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the absence and presence o f endothelial and smooth muscle cells and to characterize the role o f N O in mediating this effect. Methods: Platelet aggregation was determined turbidimetrically in platelet-rich plasma (PRP) or washed platelets from freshly sam pled venous blood from chronically cathctcrized minipigs. The for mation o f N O arising during the metabolism o f nitrates in endothe lial (EC) or smooth muscle cells (V SM C) was measured by difference spectrophotometry. Results: The superfusion o f E C or V S M C cultured on microcar rier beads with glyceryl trinitrate (G T N : 1-300 p.M. 1 min) resulted in the immediate and concentration-dependent generation o f N O . Preincubation o f P R P with G T N (0 .1-200 p.V/) for 5 min resulted in a weak concentration-dependent inhibition o f ADP-induced platelet aggregation. In the presence o f V S M C or E C the concentration response curve for G T N was significantly shifted to the left. This potentiation o f the antiaggregatory effect o f G T N was completly reversed by addition o f 10 p.V/ oxyhaemoglobin indicating that the inhibitory' effect was mediated via extracellular generation o f N O . Similar effects were obtained with isosorbide dinitrate and isosorbide 5-mononitrate. The potentiation o f the antiplatelet effect o f G T N by E C and V S M C was even more pronounced on collageninduced aggregation o f washed platelets. Pretreatment o f vascular cells with N-ethylmaleimide concentration-depcndcntly inhibited the potentiation o f the antiaggregatory effect o f nitrates suggesting the involvement o f critical sulfhydrvl groups in this process. Conclusions: (1) E C as well as V S M C metabolize organic nitrates to N O . (2) This metabolic activity appears to require the interaction with sulfhydryl groups. (3) The formation o f N O accounts for the observed potentiation o f the antiplatelet effects o f nitrates by cul tured vascular cells in vitro. (4) Besides the mere dilator component of nitrate action, an inhibitory effect on platelet aggregation might contribute substantially to the anti-ischaemic effectiveness o f these drugs and the observed beneficial effects in myocardial infarction or unstable angina.
Effect of Long-Term Isradipine Treatment on the Structure of the Endothelium in the Aorta of Spontaneously Hypertensive Rats F. Amenta. F. Ferranie, F. Abbate. E. Ciriaco Dipartimento di Sanita Pubblica c Biologia Cellulare, Univcrsita 'Tor Vergata’, Roma: Istituto di Anatomia dcgli Anim ali Domestici, Univcrsita di Messina, Italia Endothelial damage is an important factor in determining atherogcnesis. Hypertension, which causes a variety o f changes in the vas cular structure, is a factor enhancing athcrogcnesis. In view o f this we decided to investigate whether long-term treatment with the calcium antagonist isradipine was effective in protecting the endothelium from hypertension-induced damage. Ten-week male spontaneously hypertensive rats (SHR) were used. Two groups o f SH R was treated with a dose o f 0.01 mg/kg/day; or o f 0.1 mg/kg/day o f isradipine; a third group o f SH R was left untreated. Age-matched normotensivc Wistar-Kyoto (W K Y) rats were used as well. Animals were killed at 22 weeks o f age. Systolic pressure values significantly increased in control S H R . The 0.1 mg/kg dose o f isradipine but not the 0.01 rng/kg dose signifi cantly reduced systolic pressure values. The wall-to-lumcn ratio which was increased in S H R . was significantly reduced in isradipinetreated SH R . Both doses o f the compound were effective. Scanning electron microscope analysis o f the endothelium showed important structural alterations o f endothelial cells in S H R . An improved endo thelial morphology was noticeable primarily in S H R treated with the antihypertensive dose o f isradipine and in lesser amounts with the non-antihypertensive dose o f the compound. The above results suggest collectively that isradipine may have a protective effect on the endothelium o f SH R .
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Influence of Intracellular Alkalization on Endothelium-Related Vasodilation K. Ando. K. Takahashi. T. Shimosawa, A. Ono. E. Ogata, T. Fajita 4th Dept. Int. M ed., U n iv. Tokyo School M ed.. Japan T o clarify a possible role o f Na-H antiport on endotheliumrelated vasodilation possibly through changes in intracellular pH , we designated to study the effects o f changes in intracellular N a and pH in endothelium-related relaxation. Thoracic aorta was removed from 9-week-old male Sprague-Dawley rats. Aortic ring was suspended in Krebs-bicarbonate buffer aerated with 95% 02- 5 % C O : and a con traction-relaxation experiment was performed according to the stan dard procedure. In norepinephrine (3 X 10' 7 ,V/)-precontractcd aor ta. endothelium-dependent vasodilation was evaluated by cumula tive acetylcholine (ACh) vasodilation (10 _
T. F. Wischer, Basel T. Masaki, Kyoto M .J. Mulvany, Aarhus G .M . Ruhanyi, Berlin J . R. Varie, London P.M . Vanhoutte, Houston. Tex.
2nd International Symposium on Endothelium-Derived Vasoactive Factors
Editors
Basel, Switzerland, April 22-25,1992
1
2
Contractions to Endothelin-1 in Canine Coronary Arteries Are Inhibited by Sympathetic Activation
Vasa vasorum and Endothelial Cell Function in Veno-Venous Grafts
P. Aarnio, C.G.A. McGregor, V.M. Miller
G. Adamczyk, K. Lemhowicz, M. Walski, W.L. Olszewski
Department o f Surgery and Department o f Physiology and Biophysics. Mayo Clinic and Foundation. Rochester. M inn.. U SA
Surgical Research and Transplantation Laboratory, Experimental and Clinical Medical Research Centre. Polish Academy o f Sciences, and Scanning Electron Microscopy U nit. The Centre o f Child Health, Warsaw. Poland
The endothelium-derived factor endothelin-1 contracts vascular smooth muscle and inhibits release o f neurotransmitter from adren ergic and cholinergic neurons. The interaction o f these mechanisms in a blood vessel which receives both adrenergic and cholinergic innervation was studied. Canine left anterior descending coronary arteries were cut into rings, the endothelium was removed and the rings were suspended for the measurement o f isometric force in organ chambers. Endothelin-1 caused concentration-dependent con tractions in all rings. During electrical stimulation ( I Hz. 9 V . 2 ms) to activate sympathetic and parasympathetic nerve endings, the con tractions to endothelin-1 were reduced significantly. This decrease was greater in the presence o f atropine (1 X 10 6 M) and was elimi nated by a combination o f phentolamine ( 1 X 1 O' 6 M ) and propran olol ( 5 X I O *6M ). These results suggest that contractions to endothe lin-1 are reduced by transmitter released from sympathetic nerve endings. To determine the effect o f endothelin-1 presynaptically, superperfusion studies were performed. Electrical stimulation signif icantly increased release o f norepinephrine. Endothelin-1 inhibited the release o f tritium by electrical stimulation only at high concentra tions o f endothelin-1 (4 X 10"7 M). both in the absence and presence o f atropine. These results suggest that in canine coronary arteries presynaptic effects o f endothelin -1 are minimal and affect sympathetic neurotransmission. Together, the results suggest that contractions to endothelin -1 are modulated by sympathetic and parasympathetic tone postsynaptically. If these results can be generalized to human coronary arteries, then endothelin -1 may have more profound effects on vascular resistance in denervated hearts, for example, those used for transplantation.
Vasa vasorum (VV) and other structures o f the vascular wall arc damaged during harvesting the vein for grafting. This process leads to partial detachment o f endothelium (EC) and impairment o f its function. Aim o f the study was to investigate the relationship between the revascularisation o fV V o f the venous autograft and the recovery o f E C functions measured in a prostacyclin (P G L ) and fibrinolytic activity assay. The study was carried out on mongrel dogs o f both sexes weighing ==? 18 kg. The technique o f graft harvesting described by LoGcrfo was used. The study was carried out in 6 exper imental groups: ( 1 ) venovenous grafts. (2) venous grafts wrapped with plastic foil to hinder the regrowth o f V V , (3) venous grafts ligated. (4) in situ veins dissected from surrounding tissues. (5) in situ veins wrapped with plastic foil, and (6) in situ veins occluded by ligation o f both ends. Blood flow through V V was measured with use o f albumin microspheres labelled with 99mTc. Fibrinolytic activity was measured on standard fibrin plates with modified Todds tech nique. P G L production was measured in a RIA for P G F |„. Microan giograms were performed with use o f diaminobenzidine staining for endogenous peroxidase o f red cells. Scanning electron microscopy was performed. Results were expressed in percentages o f control values obtained from a nonoperated vein o f the same animal. Blood flow through VV in group 1 was 19% on day 1. 26% on day 5 and 67% on day 7. In group 2 it was 8 % on day 1.8 % on day 5, 51 % on day 7; i n group 3.3, 76 and 181 %; in group 4 30, 9 and 141 %; in group 5 21.5 and 292% and in group 6 29, 238 and 321 %. respectively. Fibrinolytic activity was in group 1 595% onday 1 .4 2 % o n d ay4 to 126% on day 7: it was
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in group 2 287, 110 and 86%; in group 3 20. 70 and 36%: in group 4 50, 231 and 155%; in group 5 34. 104 and 375%; in group 6 23. 33. and 57%, respectively. P G I 2 activity was in group 1 180% on day 1. 30% on day 4. and 123% on day 7; in group 2 it was 436, 282 and 87%; in group 3 241, 57% and 5%: in group 4 86, 137 and 43%; in group 5 320, 283 and 78%, respectively, and in group 6 1153% on day 1.2 2 6 % on day 4 and 30% on day 7. Microangiography revealed the ingrowth o f V V in group 1, on day 6. and the beginning o f their differentiation into venules and arterioles on day 7. Scanning elec tron microscopy presented full endothelial coverage in group 1 .4 , 5 and 6. The number o f E C in group I was 86% on day 6. Conclusions: Revascularisation o f V V and llow through the lumen o f venovenous grafts are necessary for maintenance o f endo thelial cell functions. Neither restoration o f flow through V V nor lumen only are satisfactory.
acetylcholine produces pulmonary vasodilation and alters V A/Q rela tionships in patients with chronic obstructive lung disease. Breathing N O induces marked and selective pulmonary vasodilation with no changes in systemic hemodynamics and no alterations in arterial oxygenation.
4
Endothelin-Converting Enzyme from Bovine and Human Endothelial Cells Kyunghye Ahn, Karen Beningo, Gregory Olds. Donald Hope Department o f Biochemistry. Pharmaceutical Research Division. Parke-Davis/Warner-Lambert Company. Ann Arbor. M ich.. U SA
Responses to Infusion of Acetylcholine and Inhalation of Nitric Oxide in Patients w ith Chronic Lung Disease and Pulmonary Hypertension 5. Adnot, C. Kouyoumdjian, M.D. Fratacci. C. Defouilloy,
P. Andrivet, S. Sediame, R. Herigaut Departement de Physiologie, IN SE R M U296, Hôpital H . Mondor, Créteil, France Endothelium-dependent relaxation in the pulmonary circulation is abolished in experimental animals during chronic exposure to hyp oxia. Moreover, isolated large pulmonary arteries o f patients with chronic lung disease show impaired relaxation to acetylcholine. We examined the responses to incremental infusion rates o f acetylcho line (5, 10 and 15 mg/min) and to inhalation o f nitric oxide (NO) at the increasing concentrations o f 5. 10. 20. and 40 ppm on hemody namics and gas exchange in 6 patients with severe chronic obstruc tive lung disease (5 m, 1 f, mean age: 56 ± 4 years). Infusion o f ace tylcholine at 15 mg/min increased cardiac index from 3.8 ± 0.4 to 4.6 ± 0.4 l/min/m2 (p < 0.01) with no change in pulmonary' artery pressure (Pap) (27 ± 1 vs. 28 ± 1 mm Hg. N S), systemic arterial pressure (Sap) ( 102 ± 3 vs. 105 ± 2 mm Hg, NS), heart rate (HR) (94 ± 5 vs. 93 ± 5 p/min, NS), cardiac filling pressures and ventilation. Pulmonary' and systemic vascular resistance decreased in a dosedependent manner, from 4.7 ± 0.4 to 3.6 ± 0.3 IU (p < 0.01 ) and from 26.5 ± 2.5 to 21 ± 5 IU (p < 0.01). respectively. Acetylcholine reduced P a 0 2 (from 56 ± 2 to 50 ± 2 mm Hg. p < 0.01 ), S a 0 2(from 85.5 ± 2.5 to 80.5 ± 2.5%, p < 0.01), S v 0 2 (from 66 ± 2 to 63 ± 2%. p < 0.05). arteriovenous 0 2 difference (from 4.1 ± 0.3 to 3.7 ± 0.3%, p < 0.05), and increased venous admixture (Q va/Q t ) (from 34.7 ± 3 to 45.7 ± 3%. p < 0.01). In contrast, breathing N O at 40 ppm decreased Pap from 29 ± 1 to 24 ± 1 mm Hg (p < 0.01 ). P V R from 4.2 ± 0.3 to 3.0 ± 0.2 IU (p < 0.01) with no other associated hemodynamic changes. Arterial P 0 2 remained unchanged or even tended to increase (from 59 ± 3 to 62 ± 3 mm Hg, NS), with a marked increase in some individual patients; Q va/Q i tended to decrease, from 34.6 ± 3.3 to 30.6 ± 3.0% (NS). We concluded that
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Potentiation of the Antiaggregatory Action of Organic Nitrates by Endothelial and Smooth Muscle Cells Is Due to Formation of Nitric Oxide ./. Aissa, M. Feelisclt Department o f Pharmacology. Schwarz Pharma. Monheim. Germany Organic nitrates exert profound vasodilator effects in vitro as well as in vivo. Former attempts to convincingly demonstrate additional antiplatelet activity in vitro, however, failed. Since cultured vascular cells have recently been demonstrated to effectively metabolize organic nitrates to N O and since N O is known to inhibit platelet aggregation, we reinvestigated the antiplatelct potential o f nitrates in
2nd Int Svmpon Endothelium-Derived Vasoactive Factors
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3
Endothelin-1 (ET-1). a highly potent vasocontractive 21-residue peptide, is formed following specific cleavage between Trp 21 and V al 22 o f the intermediate peptide ‘big E T -F (1-39 for porcine and 1-38 for human) by the putative endothclin-converting enzyme (ECE). We have identified and characterized an E C E activity from bovine and human endothelial cells. Our data clearly demonstrate that E C E is a membrane-bound metalloproteasc: substantial inhibi tion (96-98%) by metalloproteasc inhibitors (E D T A , phosphoramidon. and phenanthroline) was observed, whereas little or no inhibi tion was found for pepstatin A, leupeptin. P M SF . soybean trypsin inhibitor, or E-64. The bovine enzyme, which can be solubilized in Triton X-100. has a pH optimum o f 7.2 and a K m o f 20 \iM for big E T -1. The reaction was linear for at least 5 h. The E C E activity eluted at a volume which would correspond to a M W o f 400 kDa by gel filtration. We have elucidated the binding region o f big E T -1 for ECE and will report our progress toward the purification o f ECE. Thus. E CE is a membrane-bound metalloprotease with a putative molecular weight o f 400 kDa. Purification o f the enzyme may allow to develop specific inhibitors.
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Release of Human Umbilical Endothelium-Derived Relaxing Factor in Pregnancy-Induced Hypertension F. Akar3. M. Ark3, B.S. Hydes3. M.E. Soysalb. F SaraçoghC. N. Abacioglu3. J. van de Voordec. / Kanzik3 3Dept. Pharmacology, Fac. Pharmacy G azi U n iv ., and bAnkara Maternity Hospital, Ankara, Turkey; cLab. Normal and Path. Physiol., U niv. Ghent. Belgium The physiological abnormalities o f preeclampsia can be ex plained by dysfunction o f vascular endothelium. The present investi gation was designed to determine whether umbilical E D R F biosyn thesis differs in the normotensive pregnant and preeclamptic wom en. E D R F release from umbilical vessels (U V . donor tissues) in both groups were compared by using a cascade bioassay system. The rat aortic rings (R A R . detector tissue) were precontracted with nor adrenaline (10 ~7 M) and the EDRF-induccd relaxation in response
to histamine (HA) or bradykinin (BK) was expressed as a percentage o f precontracted tone. Addition o f H A (10 -8 —1 0 ° M ) or BK (10 -9 — 1O' 6M) to the perfusion system o f U V caused dose-dependent relax ations o f the precontracted R A R . The relaxations were inhibited with nitroarginine (I0 ~4 M) but not with indomethacin (5 X 10~5 M). In the preeclamptic group. BK-induced E D R F release was reduced significantly as compared with that o f normotensive group (57.4 ± 8.1 vs. 22.4 ± 4.2%. n = 4) in the artery and (50.4 ± 12.3 vs. 16.2 ± 5.9%, n = 4) in the vein. On the other hand. HA-induced E D R F release did not change when compared with the normotensive group (59.1 ± 8.3 vs. 39.2 ± 6.1 %. n = 5) in the artery and (56.3 ± 12.3 vs. 39.6 ± 8.2%. n = 5) in the vein. Our findings indicate a probable change in endothelial cell (EC) function but not in E C integ rity in precclampsia. Supported by N A T O grant No. CRG-900104.
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Comparison of Endothelium-Dependent Responses to Histamine of Human Gastroepiploic Artery and Saphenous Vein /•'. Akar3. B.S. Uydes3. K. AxranaoghC. A. Yenerb, S. Aflainacic. M. Arsond, A. Toriiner**. ¡Kanzik3 3Dept. Pharmacology, Fac. Pharmacy, G azi U n iv ., bDept. Cardiovascular Surgery. Fac. Medicine, G azi U n iv ., 'Turkish Organ Transplantation and Bum Foundation Hospital, and J Dept. General Surgery, Fac. Medicine, Ankara U n iv ., Ankara, Turkey The use o f gastroepiploic artery (GEA) for coronary artery' bypass grafting has been described recently. The present study was designed to compare the endothelium-dependent responses o f G E A and saphenous vein (SV) to histamine. SV and G E A were supplied by patients undergoing operation. G E A and SV rings were precon tracted with noradrenaline. SV relaxed slightly (5.3 ± 1.9%) to lower doses o f histamine ( 10 '8- 10"7 M), which disappeared with nitroar ginine (N O A R G . K H M) or denudation while it contracted to higher doses (10 -6 - 10-4 M). On the other hand, in G E A histamine ( 1O' 9- 1O' 4 M ) caused only relaxations which occurred dose-dependently (91.5 ± 6.3%). These relaxations were inhibited partially with either N O A R G . indomethacin (5 X 10“6 M) and cimetidine (10~4 M) or denudation. Histamine also caused relaxation in G E A even in the presence o f the combination o f all above antagonists (32.8 ± 10.7%). Complete inhibition was only observed with the combina tion o f the histamine receptor antagonists cimetidine and mepyramine ( 10 -5 M). These results show that in G E A histamine causes a completely different response profile than that o f SV .
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the absence and presence o f endothelial and smooth muscle cells and to characterize the role o f N O in mediating this effect. Methods: Platelet aggregation was determined turbidimetrically in platelet-rich plasma (PRP) or washed platelets from freshly sam pled venous blood from chronically cathctcrized minipigs. The for mation o f N O arising during the metabolism o f nitrates in endothe lial (EC) or smooth muscle cells (V SM C) was measured by difference spectrophotometry. Results: The superfusion o f E C or V S M C cultured on microcar rier beads with glyceryl trinitrate (G T N : 1-300 p.M. 1 min) resulted in the immediate and concentration-dependent generation o f N O . Preincubation o f P R P with G T N (0 .1-200 p.V/) for 5 min resulted in a weak concentration-dependent inhibition o f ADP-induced platelet aggregation. In the presence o f V S M C or E C the concentration response curve for G T N was significantly shifted to the left. This potentiation o f the antiaggregatory effect o f G T N was completly reversed by addition o f 10 p.V/ oxyhaemoglobin indicating that the inhibitory' effect was mediated via extracellular generation o f N O . Similar effects were obtained with isosorbide dinitrate and isosorbide 5-mononitrate. The potentiation o f the antiplatelet effect o f G T N by E C and V S M C was even more pronounced on collageninduced aggregation o f washed platelets. Pretreatment o f vascular cells with N-ethylmaleimide concentration-depcndcntly inhibited the potentiation o f the antiaggregatory effect o f nitrates suggesting the involvement o f critical sulfhydrvl groups in this process. Conclusions: (1) E C as well as V S M C metabolize organic nitrates to N O . (2) This metabolic activity appears to require the interaction with sulfhydryl groups. (3) The formation o f N O accounts for the observed potentiation o f the antiplatelet effects o f nitrates by cul tured vascular cells in vitro. (4) Besides the mere dilator component of nitrate action, an inhibitory effect on platelet aggregation might contribute substantially to the anti-ischaemic effectiveness o f these drugs and the observed beneficial effects in myocardial infarction or unstable angina.
Effect of Long-Term Isradipine Treatment on the Structure of the Endothelium in the Aorta of Spontaneously Hypertensive Rats F. Amenta. F. Ferranie, F. Abbate. E. Ciriaco Dipartimento di Sanita Pubblica c Biologia Cellulare, Univcrsita 'Tor Vergata’, Roma: Istituto di Anatomia dcgli Anim ali Domestici, Univcrsita di Messina, Italia Endothelial damage is an important factor in determining atherogcnesis. Hypertension, which causes a variety o f changes in the vas cular structure, is a factor enhancing athcrogcnesis. In view o f this we decided to investigate whether long-term treatment with the calcium antagonist isradipine was effective in protecting the endothelium from hypertension-induced damage. Ten-week male spontaneously hypertensive rats (SHR) were used. Two groups o f SH R was treated with a dose o f 0.01 mg/kg/day; or o f 0.1 mg/kg/day o f isradipine; a third group o f SH R was left untreated. Age-matched normotensivc Wistar-Kyoto (W K Y) rats were used as well. Animals were killed at 22 weeks o f age. Systolic pressure values significantly increased in control S H R . The 0.1 mg/kg dose o f isradipine but not the 0.01 rng/kg dose signifi cantly reduced systolic pressure values. The wall-to-lumcn ratio which was increased in S H R . was significantly reduced in isradipinetreated SH R . Both doses o f the compound were effective. Scanning electron microscope analysis o f the endothelium showed important structural alterations o f endothelial cells in S H R . An improved endo thelial morphology was noticeable primarily in S H R treated with the antihypertensive dose o f isradipine and in lesser amounts with the non-antihypertensive dose o f the compound. The above results suggest collectively that isradipine may have a protective effect on the endothelium o f SH R .
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Influence of Intracellular Alkalization on Endothelium-Related Vasodilation K. Ando. K. Takahashi. T. Shimosawa, A. Ono. E. Ogata, T. Fajita 4th Dept. Int. M ed., U n iv. Tokyo School M ed.. Japan T o clarify a possible role o f Na-H antiport on endotheliumrelated vasodilation possibly through changes in intracellular pH , we designated to study the effects o f changes in intracellular N a and pH in endothelium-related relaxation. Thoracic aorta was removed from 9-week-old male Sprague-Dawley rats. Aortic ring was suspended in Krebs-bicarbonate buffer aerated with 95% 02- 5 % C O : and a con traction-relaxation experiment was performed according to the stan dard procedure. In norepinephrine (3 X 10' 7 ,V/)-precontractcd aor ta. endothelium-dependent vasodilation was evaluated by cumula tive acetylcholine (ACh) vasodilation (10 _
