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Endothelium-Dependent Relaxations in Human Arteries
Endothelium-Dependent Relaxations in Human Arteries
THOMAS F. LÜSCHER, M.D.,* Department of Physiology and Biophysics; JOHN P. COOKE, M.D., Ph.D., Division of Cardiovascular Diseases and Internal Medicine; DONALD S. HOUSTON, M.D.,f Department of Physiology and Biophysics; RICARDO J. NEVES, M.D.,i Department of Surgery; PAUL M. VANHOUTTE, M.D., Department of Physiology and Biophysics Experiments were designed to study endothelium-dependent relaxations in human renal arteries (N = 13) and peripheral arteries (N = 8) suspended in organ chambers for isometric tension recording. In contracted arterial rings, acetylcholine caused endothelium-dependent relaxations that were not inhibited by indomethacin in either artery but were significantly augmented in the renal artery. Adenosine diphosphate and thrombin caused endothelium-dependent relaxations in renal but not in peripheral arteries. This finding suggests a heterogeneity of endothelium-dependent relaxations in human arteries and indicates that the relaxations are mediated by the release of an endothelium-derived relaxing factor (or factors) rather than the release of prostacyclin.
The endothelium can release vasoactive sub Data concerning endothelium-dependent relaxa stances such as prostacyclin and an endothelium- tions in human blood vessels are sparse. 13 Some derived relaxing factor (or factors). 1-4 In mam investigators who studied postmortem speci malian arteries, acetylcholine and several mens were unable to demonstrate endotheliumsubstances, including some released from aggre dependent relaxations in response to acetyl gating platelets (such as adenosine diphosphate) choline in human coronary arteries. 14 No data are or formed during coagulation (for example, throm available regarding endothelium-dependent re bin), can induce endothelium-dependent relaxa sponses to adenosine diphosphate and thrombin tions. 5-9 The latter substances might play a phys in human blood vessels. The heterogeneity of iologic role in the local control of the circulation endothelium-dependent relaxations in different and in various disease states, such as athero mammalian species and among different vas sclerosis and hypertension, in which the endo cular beds of the same species warrants the as thelium is damaged or functionally altered. 10 " 12 sessment of endothelium-dependent responses in human blood vessels. 15 " 18 The current study was performed to demonstrate endothelium*Present address: Division of Cardiology, University Hospi dependent responses in human renal and pe tal, Basel, Switzerland. ripheral arteries obtained at operation.
t Present address: University of Manitoba, Winnipeg, Manitoba, Canada. JPresent address: Oliveiro do Bairro, Portugal.
This study was supported in part by a grant from the Swiss National Foundation for the Advancement of Scientific Research. Address reprint requests to Dr. P. M. Vanhoutte, Department of Physiology and Biophysics, Mayo Clinic, Rochester, MN 55905. Mayo Clin Proc 62:601-606, 1987
METHODS Preparation of Blood Vessels and Solutions.—The experiments were performed on hu man arteries obtained intraoperatively. Renal ar teries were taken from patients undergoing nephrectomy for renal cell carcinoma (N = 12) or hydronephrosis (N = 1). Dorsalis pedis (N = 6) and 601
602
RELAXATION OF HUMAN ARTERIES
radial (N = 2) arteries were obtained from limbs after amputation for malignant bone and soft tissue tumors. The mean age of the 21 patients was 46 years (range, 13 to 74 years). The organs were obtained within minutes after they had been removed surgically. After excision, the tissue was placed into modified Krebs-Ringer bicarbonate solution (4°C) of the following composition (mM): NaCl 118.3, KC1 4.7, CaCl 2 2.5, MgS0 4 1.2, KH2PO4 1.2, N a H C 0 3 25.0, edetate calcium disodium 0.026, and glucose 11.1 (control solution). The blood vessels were cleaned of connective tissue and cut into rings (5 mm long). In some rings, the endothelium was removed by gentle rubbing with a watchmaker's forceps inserted in the lumen. The rings were mounted on stirrups and suspended in organ chambers filled with 25 ml of control solution (37°C), aerated with a mixture of 95% O2 and 5% CO2. The rings were connected to transducers (Statham Universal, UC2), and changes in isometric force were re corded. They were gradually stretched and con tracted repeatedly with norepinephrine (3 x 10"7 to 3 x 10"6 M) or prostaglandin F2« (2 x 10"6 M) until their optimal length for isometric contrac tion was reached. 19 Drugs.—The following drugs were used (all from Sigma Chemical Company, St. Louis, Mis souri): acetylcholine, adenosine diphosphate, indomethacin, L-norepinephrine bitartrate, prosta glandin F20, and thrombin (bovine). The drugs were dissolved in distilled water except for indomethacin, which was dissolved in distilled water containing Na2C03 (5 x 10~3 M) and son icated before use. The concentrations are ex pressed as final molar concentration in the bath solution. Protocols and Calculations.—Ύο study endothelium-dependent relaxations, we obtained sus tained contractions with a mean concentration of 1.6 x 10~6 M norepinephrine in renal arteries (mean increase in tension, 6.3 ± 0.3 g; this value corresponded to 45 ± 8% of the maximal contrac tion to 10~4 M norepinephrine). In peripheral arteries, contractions to norepinephrine tended to be less stable. Therefore, quantitative analysis was done only in rings contracted with prosta glandin F2a (2 x 10"6 M) (mean increase in ten sion, 5.7 ± 1.2 g). The contractions induced in rings with and without endothelium did not differ significantly. Increasing concentrations of acetylcholine (10~9 to 10 -4 M) or adenosine diphos
Mayo Clin Proc, July 1987, Vol 62
phate (10~9 to 10~4 M) were given during the contractions. Because the response to thrombin in canine blood vessels is tachyphylactic, 6 only one concentration of the substance (1 U/ml) was tested. Relaxations were expressed as percentage of the contraction to either norepinephrine or prostaglandin F20. The concentration of acetyl choline that caused 50% relaxation (IC50) was calculated for each ring. Some rings were incu bated with indomethacin (10~5 M; 30 minutes before the experiment) to inhibit the production of prostacyclin; in such cases, indomethacin was present throughout the experiment. Statistical analysis was performed with use of Student's t test for paired observations. P values smaller than 0.05 were considered statistically significant.
RESULTS Renal Arteries.—In human renal arteries con tracted with norepinephrine (1.6 x 10~6 M), acetyl choline (10~9 to 10~4 M) caused concentrationdependent relaxations in rings with, but not in those without, endothelium (N = 5; Fig. 1 and 2). The maximal relaxation averaged 81 ± 6%. The IC50 value of acetylcholine was 6.4 x 10~7 M. Indomethacin (10~5 M) significantly augmented the maximal relaxation induced by acetylcholine without significantly affecting the IC50; the in hibitor of cyclooxygenase did not significantly affect the response to norepinephrine (data not shown). In two renal arteries contracted with prostaglandin F 2 o (2 x 10 -6 M), endotheliumdependent relaxations to acetylcholine (10~9 to 10~4 M) were also present (data not shown). Adenosine diphosphate (10~9 to 10~4 M) caused concentration-dependent, endothelium-dependent relaxations (N = 5; Fig. 3). Bovine thrombin (1 U/ml) caused relaxations in rings with endothelium contracted with norepi nephrine in six of eight renal arteries (Fig. 4). In these preparations, the relaxation was preceded by a small endothelium-dependent contraction. The relaxations induced by thrombin averaged 45 ± 6%o. In two renal artery rings contracted with prostaglandin F 2a , bovine thrombin also caused endothelium-dependent relaxations. Peripheral Arteries.—In dorsalis pedis and radial arteries contracted with prostaglandin F 2a (2 x 10~6 M), acetylcholine (10~9 to 10"4 M) caused
Mayo Clin Proc, July 1987, Vol 62
RELAXATION OF HUMAN ARTERIES
Renal Artery
Dorsalis Pedis Artery
Acetylcholine, -log M
Acetylcholine, -log M
603
Endothelium: with
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Fig. 1. Isometric tension recordings in a human renal artery (left panel) and a peripheral (dorsalis pedis) artery (right panel). Arterial rings with and without endothelium were contracted with norepinephrine or prostaglandin F2tt (PGF2a). During that contraction, increasing concentrations of acetylcholine were added.
endothelium-dependent relaxations (N = 6) that were unaffected by indomethacin (N = 4; Fig. 2). The IC50 value of acetylcholine was 3.4 x 1CT7 M, and the maximal response averaged 72 ± 6%. In four peripheral arteries contracted with norepi nephrine, endothelium-dependent relaxations to acetylcholine were also present (data not shown). Adenosine diphosphate (10~9 to 10~4 M; N = 5) and bovine thrombin (1 U/ml; N = 3) did not cause endothelium-dependent relaxations in pe ripheral arteries contracted with prostaglandin F2o. Similar results were obtained in rings con tracted with norepinephrine (N = 3). DISCUSSION Acetylcholine causes endothelium-dependent re laxations in most mammalian arteries. 2-4 The present study demonstrates that human renal and peripheral arteries also exhibit potent and comparable endothelium-dependent relaxations in response to acetylcholine. Blockade of cyclo-
oxygenase by indomethacin did not inhibit these relaxations; this finding suggests that acetyl choline does not cause relaxation by release of vasodilator prostanoids such as prostacyclin. Hence, a likely assumption is that it causes re lease of "an endothelium-derived relaxing fac tor (or factors)" 2 " 4 in these preparations. In renal arteries, indomethacin enhanced the endothelium-dependent relaxations to acetyl choline at higher concentrations. Similar results have been obtained in human mesenteric arteries in response to bradykinin. 13 Adenosine diphosphate is released from aggre gating human platelets. 16 The substance induces endothelium-dependent relaxations in canine coronary and femoral arteries and in the rat aorta. 3,5,9,10 In canine coronary arteries, adeno sine diphosphate released from canine or human platelets is the major mediator of the relaxations observed in vascular rings with endothelium dur ing platelet aggregation. 9,12,16 Thrombin likewise induces endothelium-dependent relaxations in a
604
RELAXATION OF HUMAN ARTERIES
Mayo Clin Proc, July 1987, Vol 62
Renal Arteries
Peripheral Arteries
■ = with endothelium
* = without endothelium
o = w i t h endothelium
o = without endothelium
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Acetylcholine, - l o g M Fig. 2. Endothelium-dependent relaxations induced by acetylcholine in human renal arteries (left panel ) and peripheral arteries (right panel ) under control conditions or after incubation with indomethacin (10~5 M). Rings with and without endothelium were contracted with norepinephrine (renal arteries) or prostaglandin F2l, (peripheral arteries). Results are expressed as percentage of that contraction. Data are presented as means + SEM. Asterisks denote that difference between rings with endothelium in the presence and absence of indomethacin is statistically significant (P<0.05).
variety of canine blood vessels.3,5,6 In the current study, adenosine diphosphate and thrombin both induced endothelium-dependent relaxations in human renal but not peripheral arteries. These results suggest that, in humans, the endothelium of certain arteries may contribute to vascular responses during platelet aggregation or activa tion of the coagulation cascade. This relationship did not exist in the human peripheral arteries studied inasmuch as they did not exhibit endothelium-dependent relaxations to adenosine diphosphate or thrombin. This difference of endothelium-dependent responses in human re nal and peripheral arteries was independent of the agonist (that is, norepinephrine or prosta glandin F20) used to contract the rings. The re laxations to acetylcholine were comparable in
renal and peripheral arteries. Although no sys tematic morphologic analysis was performed, this finding suggests that functional endothe lium was present in both types of preparations. Hence, the observed differences in the response to adenosine diphosphate and thrombin between human renal and peripheral arteries likely rep resent a heterogeneity of endothelium-dependent vascular responses in humans similar to that demonstrated in dogs.5,15,17,18 The fact that the peripheral and renal arteries used in this study were obtained from different patients may some what limit this interpretation. The qualitative rather than the quantitative difference of endothelium-dependent responses in these two vascular beds, however, makes this explanation unlikely.
Mayo Clin Proc, July 1987, Vol 62
RELAXATION OF HUMAN ARTERIES
605
CONCLUSION This study demonstrates that endotheliumdependent relaxations can be evoked in human arteries and that these relaxations probably are due to the release of an endothelium-derived re laxing factor (or factors). The study further sug gests a heterogeneity of endothelium-dependent responses in the renal and peripheral vascular beds.
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ACKNOWLEDGMENT We thank Robert R. Lorenz and Helen I. Hen drickson for preparing the illustrations and Janet M. Beckman for typing the original manuscript.
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REFERENCES 9
8
7
6
5
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Adenosine Diphosphate, -log M
Fig. 3. Endothelium-dependent relaxations induced by aden osine diphosphate in human renal arteries. Arterial rings with and without endothelium were contracted with norepinephrine. Results are expressed as percentage of that con traction. Data are presented as means + SEM. Asterisks denote that difference between rings with and without endo thelium is statistically significant (P<0.05).
With endothelium
Thrombin, 1U/ml \
5 min Without endothelium
Thrombin, 1U/ml
Norepinephrine, 10"7M
Fig. 4. Endothelium-dependent relaxation induced by throm bin in a human renal artery. Arterial rings with and without endothelium were contracted with norepinephrine (1(Γ7 Μ). Bovine thrombin (1 U/ml) was added during that contraction.
1. Moncada S, Vane JR: Prostacyclin (PGI2), the vascular wall and vasodilatation. In Mechanisms of Vasodilatation. Edited by PM Vanhoutte, I Leusen. Basel, Swit zerland, S Karger, 1978, pp 107-121 2. Furchgott RF, Zawadzki JV: The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288:373-376, 1980 3. Furchgott RF: Role of endothelium in responses of vas cular smooth muscle. Circ Res 53:557-573, 1983 4. Vanhoutte PM, Rubanyi GM, Miller VM, Houston DS: Modulation of vascular smooth muscle contraction by the endothelium. Ann Rev Physiol 48:307-320, 1986 5. De Mey JG, Vanhoutte PM: Heterogeneous behavior of the canine arterial and venous wall: importance of the endothelium. Circ Res 51:439-447, 1982 6. De Mey JG, Claeys M, Vanhoutte PM: Endotheliumdependent inhibitory effects of acetylcholine, adenosine triphosphate, thrombin and arachidonic acid in the canine femoral artery. J Pharmacol Exp Ther 222:166173, 1982 7. Cohen RA, Shepherd JT, Vanhoutte PM: Inhibitory role of the endothelium in the response of isolated coronary arteries to platelets. Science 221:273-274, 1983 8. Cohen RA, Shepherd JT, Vanhoutte PM: Endothelium and asymmetrical responses of the coronary arterial wall. Am J Physiol 247:H403-H408, 1984 9. Houston DS, Shepherd JT, Vanhoutte PM: Adenine nucleotides, serotonin, and endothelium-dependent relaxa tions to platelets. Am J Physiol 248:H389-H395,1985 10. Lüscher TF, Vanhoutte PM: Endothelium-dependent contractions to acetylcholine in the aorta of the spon taneously hypertensive rat. Hypertension 8:344-348,1986 11. Lüscher TF, Raij L, Vanhoutte PM: Decreased endothelium-dependent relaxations to acetylcholine in the aorta of hypertensive Dahl rats (abstract). J Am Coll Cardiol 7 (Suppl A):245A, 1986 12. Vanhoutte PM, Houston DS: Platelets, endothelium, and vasospasm. Circulation 72:728-734,1985 13. Cherry PD, Furchgott RF, Zawadzki JV, Jothianandan D: Role of endothelial cells in relaxation of isolated arteries by bradykinin. Proc Natl Acad Sei USA 79:21062110,1982
606
RELAXATION OF HUMAN ARTERIES
14. Kalsner S: Cholinergic mechanisms in human coronary artery preparations: implications of species differences. J Physiol 358:509-526, 1985 15. Vanhoutte PM, Miller VM: Heterogeneity of endotheliumdependent responses in mammalian blood vessels. J Cardiovasc Pharmacol 7 (Suppl 3):S12-S22,1985 16. Houston DS, Vanhoutte PM: Direct contraction and endothelium-mediated relaxation of isolated canine coronary arteries by aggregating human platelets (ab stract). Fed Proc 44:1562, 1985
Mayo Clin Proc, July 1987, Vol 62
17. Houston DS, Shepherd JT, Vanhoutte PM: Platelets cause endothelium-dependent relaxations in some but not all isolated canine vessels (abstract). Fed Proc 43:1084, 1984 18. Katusic ZS, Shepherd JT, Vanhoutte PM: Vasopressin causes endothelium-dependent relaxations of the canine basilar artery. Circ Res 55:575-579,1984 19. Vanhoutte P, Leusen I: The reactivity of isolated venous preparations to electrical stimulation. Pflugers Arch 306:341-353,1969
THOMAS F. LÜSCHER, M.D.,* Department of Physiology and Biophysics; JOHN P. COOKE, M.D., Ph.D., Division of Cardiovascular Diseases and Internal Medicine; DONALD S. HOUSTON, M.D.,f Department of Physiology and Biophysics; RICARDO J. NEVES, M.D.,i Department of Surgery; PAUL M. VANHOUTTE, M.D., Department of Physiology and Biophysics Experiments were designed to study endothelium-dependent relaxations in human renal arteries (N = 13) and peripheral arteries (N = 8) suspended in organ chambers for isometric tension recording. In contracted arterial rings, acetylcholine caused endothelium-dependent relaxations that were not inhibited by indomethacin in either artery but were significantly augmented in the renal artery. Adenosine diphosphate and thrombin caused endothelium-dependent relaxations in renal but not in peripheral arteries. This finding suggests a heterogeneity of endothelium-dependent relaxations in human arteries and indicates that the relaxations are mediated by the release of an endothelium-derived relaxing factor (or factors) rather than the release of prostacyclin.
The endothelium can release vasoactive sub Data concerning endothelium-dependent relaxa stances such as prostacyclin and an endothelium- tions in human blood vessels are sparse. 13 Some derived relaxing factor (or factors). 1-4 In mam investigators who studied postmortem speci malian arteries, acetylcholine and several mens were unable to demonstrate endotheliumsubstances, including some released from aggre dependent relaxations in response to acetyl gating platelets (such as adenosine diphosphate) choline in human coronary arteries. 14 No data are or formed during coagulation (for example, throm available regarding endothelium-dependent re bin), can induce endothelium-dependent relaxa sponses to adenosine diphosphate and thrombin tions. 5-9 The latter substances might play a phys in human blood vessels. The heterogeneity of iologic role in the local control of the circulation endothelium-dependent relaxations in different and in various disease states, such as athero mammalian species and among different vas sclerosis and hypertension, in which the endo cular beds of the same species warrants the as thelium is damaged or functionally altered. 10 " 12 sessment of endothelium-dependent responses in human blood vessels. 15 " 18 The current study was performed to demonstrate endothelium*Present address: Division of Cardiology, University Hospi dependent responses in human renal and pe tal, Basel, Switzerland. ripheral arteries obtained at operation.
t Present address: University of Manitoba, Winnipeg, Manitoba, Canada. JPresent address: Oliveiro do Bairro, Portugal.
This study was supported in part by a grant from the Swiss National Foundation for the Advancement of Scientific Research. Address reprint requests to Dr. P. M. Vanhoutte, Department of Physiology and Biophysics, Mayo Clinic, Rochester, MN 55905. Mayo Clin Proc 62:601-606, 1987
METHODS Preparation of Blood Vessels and Solutions.—The experiments were performed on hu man arteries obtained intraoperatively. Renal ar teries were taken from patients undergoing nephrectomy for renal cell carcinoma (N = 12) or hydronephrosis (N = 1). Dorsalis pedis (N = 6) and 601
602
RELAXATION OF HUMAN ARTERIES
radial (N = 2) arteries were obtained from limbs after amputation for malignant bone and soft tissue tumors. The mean age of the 21 patients was 46 years (range, 13 to 74 years). The organs were obtained within minutes after they had been removed surgically. After excision, the tissue was placed into modified Krebs-Ringer bicarbonate solution (4°C) of the following composition (mM): NaCl 118.3, KC1 4.7, CaCl 2 2.5, MgS0 4 1.2, KH2PO4 1.2, N a H C 0 3 25.0, edetate calcium disodium 0.026, and glucose 11.1 (control solution). The blood vessels were cleaned of connective tissue and cut into rings (5 mm long). In some rings, the endothelium was removed by gentle rubbing with a watchmaker's forceps inserted in the lumen. The rings were mounted on stirrups and suspended in organ chambers filled with 25 ml of control solution (37°C), aerated with a mixture of 95% O2 and 5% CO2. The rings were connected to transducers (Statham Universal, UC2), and changes in isometric force were re corded. They were gradually stretched and con tracted repeatedly with norepinephrine (3 x 10"7 to 3 x 10"6 M) or prostaglandin F2« (2 x 10"6 M) until their optimal length for isometric contrac tion was reached. 19 Drugs.—The following drugs were used (all from Sigma Chemical Company, St. Louis, Mis souri): acetylcholine, adenosine diphosphate, indomethacin, L-norepinephrine bitartrate, prosta glandin F20, and thrombin (bovine). The drugs were dissolved in distilled water except for indomethacin, which was dissolved in distilled water containing Na2C03 (5 x 10~3 M) and son icated before use. The concentrations are ex pressed as final molar concentration in the bath solution. Protocols and Calculations.—Ύο study endothelium-dependent relaxations, we obtained sus tained contractions with a mean concentration of 1.6 x 10~6 M norepinephrine in renal arteries (mean increase in tension, 6.3 ± 0.3 g; this value corresponded to 45 ± 8% of the maximal contrac tion to 10~4 M norepinephrine). In peripheral arteries, contractions to norepinephrine tended to be less stable. Therefore, quantitative analysis was done only in rings contracted with prosta glandin F2a (2 x 10"6 M) (mean increase in ten sion, 5.7 ± 1.2 g). The contractions induced in rings with and without endothelium did not differ significantly. Increasing concentrations of acetylcholine (10~9 to 10 -4 M) or adenosine diphos
Mayo Clin Proc, July 1987, Vol 62
phate (10~9 to 10~4 M) were given during the contractions. Because the response to thrombin in canine blood vessels is tachyphylactic, 6 only one concentration of the substance (1 U/ml) was tested. Relaxations were expressed as percentage of the contraction to either norepinephrine or prostaglandin F20. The concentration of acetyl choline that caused 50% relaxation (IC50) was calculated for each ring. Some rings were incu bated with indomethacin (10~5 M; 30 minutes before the experiment) to inhibit the production of prostacyclin; in such cases, indomethacin was present throughout the experiment. Statistical analysis was performed with use of Student's t test for paired observations. P values smaller than 0.05 were considered statistically significant.
RESULTS Renal Arteries.—In human renal arteries con tracted with norepinephrine (1.6 x 10~6 M), acetyl choline (10~9 to 10~4 M) caused concentrationdependent relaxations in rings with, but not in those without, endothelium (N = 5; Fig. 1 and 2). The maximal relaxation averaged 81 ± 6%. The IC50 value of acetylcholine was 6.4 x 10~7 M. Indomethacin (10~5 M) significantly augmented the maximal relaxation induced by acetylcholine without significantly affecting the IC50; the in hibitor of cyclooxygenase did not significantly affect the response to norepinephrine (data not shown). In two renal arteries contracted with prostaglandin F 2 o (2 x 10 -6 M), endotheliumdependent relaxations to acetylcholine (10~9 to 10~4 M) were also present (data not shown). Adenosine diphosphate (10~9 to 10~4 M) caused concentration-dependent, endothelium-dependent relaxations (N = 5; Fig. 3). Bovine thrombin (1 U/ml) caused relaxations in rings with endothelium contracted with norepi nephrine in six of eight renal arteries (Fig. 4). In these preparations, the relaxation was preceded by a small endothelium-dependent contraction. The relaxations induced by thrombin averaged 45 ± 6%o. In two renal artery rings contracted with prostaglandin F 2a , bovine thrombin also caused endothelium-dependent relaxations. Peripheral Arteries.—In dorsalis pedis and radial arteries contracted with prostaglandin F 2a (2 x 10~6 M), acetylcholine (10~9 to 10"4 M) caused
Mayo Clin Proc, July 1987, Vol 62
RELAXATION OF HUMAN ARTERIES
Renal Artery
Dorsalis Pedis Artery
Acetylcholine, -log M
Acetylcholine, -log M
603
Endothelium: with
98
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Norepinephrine, 3x10" 7 M
9
8
7
7 6 5 4
1
5 min
Fig. 1. Isometric tension recordings in a human renal artery (left panel) and a peripheral (dorsalis pedis) artery (right panel). Arterial rings with and without endothelium were contracted with norepinephrine or prostaglandin F2tt (PGF2a). During that contraction, increasing concentrations of acetylcholine were added.
endothelium-dependent relaxations (N = 6) that were unaffected by indomethacin (N = 4; Fig. 2). The IC50 value of acetylcholine was 3.4 x 1CT7 M, and the maximal response averaged 72 ± 6%. In four peripheral arteries contracted with norepi nephrine, endothelium-dependent relaxations to acetylcholine were also present (data not shown). Adenosine diphosphate (10~9 to 10~4 M; N = 5) and bovine thrombin (1 U/ml; N = 3) did not cause endothelium-dependent relaxations in pe ripheral arteries contracted with prostaglandin F2o. Similar results were obtained in rings con tracted with norepinephrine (N = 3). DISCUSSION Acetylcholine causes endothelium-dependent re laxations in most mammalian arteries. 2-4 The present study demonstrates that human renal and peripheral arteries also exhibit potent and comparable endothelium-dependent relaxations in response to acetylcholine. Blockade of cyclo-
oxygenase by indomethacin did not inhibit these relaxations; this finding suggests that acetyl choline does not cause relaxation by release of vasodilator prostanoids such as prostacyclin. Hence, a likely assumption is that it causes re lease of "an endothelium-derived relaxing fac tor (or factors)" 2 " 4 in these preparations. In renal arteries, indomethacin enhanced the endothelium-dependent relaxations to acetyl choline at higher concentrations. Similar results have been obtained in human mesenteric arteries in response to bradykinin. 13 Adenosine diphosphate is released from aggre gating human platelets. 16 The substance induces endothelium-dependent relaxations in canine coronary and femoral arteries and in the rat aorta. 3,5,9,10 In canine coronary arteries, adeno sine diphosphate released from canine or human platelets is the major mediator of the relaxations observed in vascular rings with endothelium dur ing platelet aggregation. 9,12,16 Thrombin likewise induces endothelium-dependent relaxations in a
604
RELAXATION OF HUMAN ARTERIES
Mayo Clin Proc, July 1987, Vol 62
Renal Arteries
Peripheral Arteries
■ = with endothelium
* = without endothelium
o = w i t h endothelium
o = without endothelium
I n d o m e t h a c i n , 10"°M
I n d o m e t h a c i n , 10"°M
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Acetylcholine, - l o g M Fig. 2. Endothelium-dependent relaxations induced by acetylcholine in human renal arteries (left panel ) and peripheral arteries (right panel ) under control conditions or after incubation with indomethacin (10~5 M). Rings with and without endothelium were contracted with norepinephrine (renal arteries) or prostaglandin F2l, (peripheral arteries). Results are expressed as percentage of that contraction. Data are presented as means + SEM. Asterisks denote that difference between rings with endothelium in the presence and absence of indomethacin is statistically significant (P<0.05).
variety of canine blood vessels.3,5,6 In the current study, adenosine diphosphate and thrombin both induced endothelium-dependent relaxations in human renal but not peripheral arteries. These results suggest that, in humans, the endothelium of certain arteries may contribute to vascular responses during platelet aggregation or activa tion of the coagulation cascade. This relationship did not exist in the human peripheral arteries studied inasmuch as they did not exhibit endothelium-dependent relaxations to adenosine diphosphate or thrombin. This difference of endothelium-dependent responses in human re nal and peripheral arteries was independent of the agonist (that is, norepinephrine or prosta glandin F20) used to contract the rings. The re laxations to acetylcholine were comparable in
renal and peripheral arteries. Although no sys tematic morphologic analysis was performed, this finding suggests that functional endothe lium was present in both types of preparations. Hence, the observed differences in the response to adenosine diphosphate and thrombin between human renal and peripheral arteries likely rep resent a heterogeneity of endothelium-dependent vascular responses in humans similar to that demonstrated in dogs.5,15,17,18 The fact that the peripheral and renal arteries used in this study were obtained from different patients may some what limit this interpretation. The qualitative rather than the quantitative difference of endothelium-dependent responses in these two vascular beds, however, makes this explanation unlikely.
Mayo Clin Proc, July 1987, Vol 62
RELAXATION OF HUMAN ARTERIES
605
CONCLUSION This study demonstrates that endotheliumdependent relaxations can be evoked in human arteries and that these relaxations probably are due to the release of an endothelium-derived re laxing factor (or factors). The study further sug gests a heterogeneity of endothelium-dependent responses in the renal and peripheral vascular beds.
n=5 120r
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c
Q.
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ACKNOWLEDGMENT We thank Robert R. Lorenz and Helen I. Hen drickson for preparing the illustrations and Janet M. Beckman for typing the original manuscript.
*-
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u
CO
J: c o o »*o
40
20
■ •with endothelium ♦-without endothelium
REFERENCES 9
8
7
6
5
4
Adenosine Diphosphate, -log M
Fig. 3. Endothelium-dependent relaxations induced by aden osine diphosphate in human renal arteries. Arterial rings with and without endothelium were contracted with norepinephrine. Results are expressed as percentage of that con traction. Data are presented as means + SEM. Asterisks denote that difference between rings with and without endo thelium is statistically significant (P<0.05).
With endothelium
Thrombin, 1U/ml \
5 min Without endothelium
Thrombin, 1U/ml
Norepinephrine, 10"7M
Fig. 4. Endothelium-dependent relaxation induced by throm bin in a human renal artery. Arterial rings with and without endothelium were contracted with norepinephrine (1(Γ7 Μ). Bovine thrombin (1 U/ml) was added during that contraction.
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