T. F. Lüscher

Both ETA and ETB receptors mediate contraction to endothelin-1 in human blood vessels.

BACKGROUNDnEndothelin (ET)-1 has potent vascular effects. Two endothelin receptors have been cloned, namely, the ETA receptor, which preferentially binds ET-1, and the ETB receptor, which equally binds ET-1 and ET-3 and preferentially sarafotoxin S6c. We characterized endothelin receptor subtypes on vascular smooth muscle and endothelium of isolated human internal mammary artery (IMA) and vein (IMV) and porcine coronary artery (PCA) using the ETA antagonists FR139317 and BQ-123, the ETB ligand sarafotoxin S6c, and the ETA/ETB antagonist Ro 47-0203 (bosentan).nnnMETHODS AND RESULTSnIn endothelium-denuded IMA and PCA and less so in IMV, FR139317 and BQ-123 (in PCA only) shifted the concentration-contraction curves to ET-1 parallel to the right. However, even at 10(-5) mol/L, FR139317 did not inhibit a high-sensitivity portion of the concentration-contraction curve. Moreover, the ETB receptor agonist sarafotoxin S6c induced contraction in vessels preincubated with FR139317. IMV was significantly more sensitive to the contractile effect of ET-1 and sarafotoxin S6c than was IMA (P < .05). Prolonged incubation with sarafotoxin S6c (to downregulate ETB receptors) and FR139317 eliminated the contraction resistant to FR139317. The ETA/ETB receptor antagonist bosentan caused a parallel shift of the concentration-contraction curve to the right at all concentrations of endothelin. ETB receptor mRNA was detected by Northern blot analysis in IMA and aortic smooth muscle cells. In precontracted IMA and PCA with endothelium, sarafotoxin S6c did not cause endothelium-dependent relaxations, whereas transient responses occurred in IMV.nnnCONCLUSIONSnVascular smooth muscle cells of human IMA, IMV, and PCA contain both ETA and ETB receptors, whereas the endothelium of IMA and PCA does not express functional ETB receptors linked to nitric oxide and/or prostacyclin production. Hence, inhibition of endothelin-induced contraction in patients requires the use of combined ETA/ETB antagonists.

Threshold concentrations of endothelin-1 potentiate contractions to norepinephrine and serotonin in human arteries. A new mechanism of vasospasm?

Endothelin-1 is an endothelium-derived vasoconstrictor peptide. Its circulating levels are below those known to evoke direct vascular effects. To study whether low concentrations of endothelin-1 potentiate the effects of other vasoconstrictor hormones, we suspended isolated human internal mammary and left anterior descending coronary artery rings in organ chambers for isometric tension recording. In mammary artery rings, the contractions to norepinephrine (3 x 10(-8) M) were potentiated by threshold (3 x 10(-10) M) and low concentrations (10(-9) M) of endothelin-1 (96 +/- 35% and 149 +/- 58% increase from control; p less than 0.01 and 0.001; n = 6). The inhibitor of endothelial nitric oxide formation L-NG-monomethyl arginine did not affect the potentiating effects of the peptide. The calcium antagonist darodipine (10(-7) M) prevented the potentiation of the response to norepinephrine evoked by endothelin-1. Similarly, contractions to serotonin (10(-7) or 3 x 10(-8) M) were amplified by endothelin-1 (3 x 10(-10) M) in the mammary (30 +/- 9%) and in the coronary arteries (59 +/- 25%). Endothelin-1 (10(-9) M) further potentiated the response (57 +/- 23% in mammary and 87 +/- 26% in coronary arteries; p less than 0.05; n = 7 and 3). The sensitivity of mammary arteries to calcium chloride was markedly enhanced in the presence of endothelin-1 (3 x 10(-10) M; concentration shift, eightfold; p less than 0.01; n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidized low density lipoproteins inhibit relaxations of porcine coronary arteries. Role of scavenger receptor and endothelium-derived nitric oxide.

BACKGROUNDnWe studied the effects of low density lipoprotein (LDL) on endothelium function.nnnMETHODS AND RESULTSnPorcine epicardial and intramyocardial coronary arteries suspended in organ chambers for isometric tension recording were exposed to LDL for 2 hours and were then washed. In epicardial coronary arteries, oxidized LDL (30-300 micrograms/ml) but not native LDL or lysolecithin inhibited endothelium-dependent relaxations to serotonin, thrombin, and aggregating platelets (5,000-75,000/microliter). Endothelium-dependent relaxations to bradykinin and A23187 and endothelium-independent relaxations to SIN-1 were unaffected by oxidized LDL. In intramyocardial coronary arteries, oxidized LDL had no appreciable effect on relaxations to serotonin. The effect of oxidized LDL on the response to serotonin in epicardial coronary arteries was completely prevented by dextran sulfate (10 micrograms/ml). The inhibitory effect of oxidized LDL persisted in the presence of pertussis toxin. Similar to the lipoproteins, L-NG-monomethyl arginine (L-NMMA) reduced relaxations to serotonin but not to bradykinin in epicardial coronary arteries. In the presence of L-NMMA, oxidized LDL further reduced the response to serotonin. In arteries in which relaxations to serotonin were inhibited by oxidized LDL, L-arginine but not D-arginine induced a full relaxation. Pretreatment with L-arginine potentiated relaxations to serotonin in arteries exposed to oxidized LDL.nnnCONCLUSIONSnThus, oxidized LDL activates the scavenger receptor on endothelial cells and inhibits the receptor-operated nitric oxide formation in epicardial but not in intramyocardial coronary arteries. The mechanism is not related to dysfunction of a Gi protein but is related to a reduced intracellular availability of L-arginine. The reduced nitric oxide formation at sites of early atherosclerotic lesions may favor platelet aggregation and vasospasm, both of which are known clinical events in patients with coronary artery disease.

Interaction between endothelin-1 and endothelium-derived relaxing factor in human arteries and veins.

Endothelin-1 is a 21-amino acid endothelial vasoconstrictor peptide that may be the physiological antagonist of endothelium-derived relaxing factor (EDRF). Endothelin-1 (10(-11)-3 x 10(-7) M) evoked potent contractions of isolated internal mammary arteries, internal mammary veins, and saphenous veins, which were enhanced in internal mammary veins as compared with internal mammary arteries (concentration shift, 6.3-fold; p less than 0.05) but not in the saphenous veins. Endothelial removal augmented the response to the peptide (at 3 x 10(-7) M) in internal mammary arteries (p less than 0.05) but not in veins. In the artery, EDRF released by acetylcholine or bradykinin reversed endothelin-1-induced contractions; in saphenous veins, both agonists were much less effective compared with the artery and veins contracted with norepinephrine (p less than 0.005-0.01). This inhibition of endothelium-dependent relaxations in veins occurred at half-maximal contractions but was most prominent at maximal contractions to the peptide. Nitric oxide similarly inhibited contractions to endothelin-1 and norepinephrine in internal mammary arteries, whereas in veins that were contracted with endothelin-1 but not with norepinephrine, the relaxations were blunted (p less than 0.005). The nitric oxide donor SIN-1 and sodium nitroprusside induced complete relaxations of internal mammary arteries but were less effective in veins contracted with endothelin-1 (p less than 0.005). Thus, in normal human arteries, EDRF inhibits endothelin-1-induced contractions, whereas the peptide specifically attenuates the effects of EDRF and nitrovasodilators in veins. This may be important in pathological conditions associated with increased levels of endothelin-1 and in veins used as coronary bypass grafts.

Different activation of the endothelial L-arginine and cyclooxygenase pathway in the human internal mammary artery and saphenous vein.

The endothelium releases substances controlling vascular tone and platelet function. We investigated mediators of endothelium-dependent responses in human internal mammary arteries and saphenous veins. The inhibitor of nitric oxide formation, NG-monomethyl L-arginine, enhanced the sensitivity to norepinephrine (fivefold) and evoked more pronounced endothelium-dependent contractions in internal mammary arteries (19 +/- 6% of 100 mM KCl) than in saphenous veins (2 +/- 1%; p less than 0.005). In internal mammary arteries, NG-monomethyl L-arginine, but not indomethacin, markedly reduced endothelium-dependent relaxations to acetylcholine (from 95 +/- 2% to 39 +/- 7%; p less than 0.005) and prevented those to histamine (78 +/- 6% to 4 +/- 3%; p less than 0.005). In saphenous veins, endothelium-dependent relaxations to acetylcholine were weak (24 +/- 11%), while nitric oxide caused comparable relaxations (85 +/- 3%) as in internal mammary arteries (80 +/- 5%; NS). NG-Monomethyl L-arginine prevented the relaxations to acetylcholine and unmasked endothelium-dependent contractions (30 +/- 10%). Indomethacin and the thromboxane synthetase inhibitor CGS-13080 augmented relaxations of saphenous veins to acetylcholine from 24 +/- 11% to 46 +/- 9% (p less than 0.05). Histamine-evoked contractions were converted to endothelium-dependent relaxations by indomethacin and the thromboxane A2/endoperoxide receptor antagonist SQ-30741 (38 +/- 3% and 40 +/- 6%; p less than 0.05) but not CGS-13080. Thus, 1) nitric oxide mediates endothelium-dependent relaxations in human arteries and veins; 2) internal mammary arteries release more nitric oxide than do saphenous veins, and 3) in saphenous veins, the effects of nitric oxide are reduced by endothelium-derived contracting factors originating from the cyclooxygenase pathway.

Implications of pulsatile stretch on growth of saphenous vein and mammary artery smooth muscle

Internal mammary artery (IMA) coronary bypass grafts have a higher patency than saphenous vein (SV) grafts. Intimal hyperplasia and occlusion of venous grafts result from smooth muscle proliferation. Mechanical factors, such as pulsatile stretch, are potential mediators of this process. Smooth muscle cells from IMA and SV were cultured on deformable membranes and exposed to pulsatile stretch (60 cycles/min). This stimulus increased 3H-thymidine incorporation into venous (a two-fold increase) but not arterial smooth muscle cells after 24 h. Smooth muscle cell numbers from SV, but not IMA, were increased (p less than 0.05) after 6 days of stretch. Thus, pulsatile stretch stimulates smooth muscle cell proliferation in SV, but not IMA, and may contribute to venous bypass graft disease.

Calcium antagonists differently inhibit proliferation of human coronary smooth muscle cells in response to pulsatile stretch and platelet-derived growth factor.

BACKGROUNDnVascular smooth muscle cell proliferation is the key event of coronary artery disease. Mechanical forces, in particular, pulsatile stretch and platelet-derived growth factor, may play an important role.nnnMETHODS AND RESULTSnVascular smooth muscle cells were cultured from the media of human left descending coronary arteries obtained from organ donors using the explant method. To study effects of pulsatile stretch on vascular smooth muscle cell proliferation, a computer-controlled in vitro pulsatile stretch device was used. Cells were seeded onto Flex I culture plates with deformable membranes and exposed to pulsatile stretch (60 cycles per minute) and/or growth factors. Proliferation of smooth muscle cells was determined by 3H-thymidine incorporation. Pulsatile stretch markedly stimulated 3H-thymidine incorporation of coronary smooth muscle cells (180 +/- 15 to 432 +/- 27 cpm/10(5) cells; P < .05) after 24 hours and increased cell number after 6 days (10.3 +/- 0.7 x 10(4)/mL; P < .05). Platelet-derived growth factor-AB (0.01 to 10 ng/mL) concentration-dependently stimulated 3H-thymidine incorporation in coronary smooth muscle cells (EC50, 0.1 ng/mL) and had additive effects with pulsatile stretch. The Ca2+ antagonist verapamil (10(-7) to 10(-5) M) concentration-dependently inhibited proliferation stimulated by platelet-derived growth factor back to control levels (P < .05 to .01) but not that induced by pulsatile stretch.nnnCONCLUSIONSnPulsatile stretch and platelet-derived growth factor are potent stimuli for proliferation of coronary smooth muscle cells. The selective inhibitory effect of a Ca2+ antagonist on smooth muscle cell proliferation stimulated by platelet-derived growth factor but not by pulsatile stretch may explain why the drugs have only modest antiatherogenic effects in patients with coronary artery disease.

Endothelium-derived relaxing factor and protection against contractions induced by histamine and serotonin in the human internal mammary artery and in the saphenous vein.

We investigated the release of endothelium-derived relaxing factor (EDRF) in response to serotonin and histamine in the human internal mammary artery and saphenous vein. The arteries and veins were obtained intraoperatively and were suspended in organ chambers to record isometric tension. In mammary arteries, histamine (10(-8) to 3 X 10(-6) M) induced relaxations in rings with (70 +/- 5%, IC50, 6.5 +/- 0.2) but not without endothelium (p less than 0.005 for rings with compared with those without endothelium, n = 7-10). The response was inhibited by methylene blue or hemoglobin, but not meclofenamate, and, therefore, EDRF was delineated as the mediator. Because chlorpheniramine but not cimetidine inhibited the response, EDRF was released by the H1-histaminergic receptor (n = 5-8). In contrast, in saphenous veins, histamine caused only weak or absent endothelium-dependent relaxations, but contractions were enhanced in rings with endothelium (p less than 0.05, n = 6). Serotonin did not induce endothelium-dependent relaxations, but contractions were markedly greater in veins compared with arteries (p less than 0.005, n = 6). The endothelium inhibited the maximal contraction to serotonin in arteries (p less than 0.034) but not in veins. Thus, EDRF protects against contractions induced by histamine and serotonin in the mammary artery but not in the saphenous vein. This may be important for improved graft function and patency of the artery compared with that of the vein.

Different interactions of platelets with arterial and venous coronary bypass vessels

We studied the interaction of platelets with the wall of human internal mammary arteries and saphenous veins, suspended in organ chambers for measurement of isometric tension. Vessels were obtained during coronary bypass surgery and cut into 5 mm rings; in some the endothelium was chemically removed. Rings from several patients were randomly chosen for each experiment, and in each ring six concentrations of platelets (from healthy blood donors; 1-75 x 10(3)/microliters) were tested consecutively and concentration-response curves constructed; the areas under these curves were used for statistical comparisons. In rings of internal mammary artery contracted in response to noradrenaline, aggregating platelets induced endothelium-dependent relaxation which was prevented by apyrase (0.67 U/ml ADPase activity) and L-NG-monomethylarginine (1 mmol/l). By contrast, in saphenous vein rings contracted in response to noradrenaline, aggregating platelets induced only a further increase in tension. In quiescent vessels, the platelet-induced contraction did not occur in arteries with endothelium but that in veins was greater and facilitated by endothelium. Preincubation of platelets with aspirin (10 mumol/l) reduced the contraction in both vessels, but contraction was abolished only in the presence of both the thromboxane receptor antagonist SQ-30741 and the serotoninergic (5HT2) receptor antagonist ketanserin. These findings show that platelet-derived ADP causes release of nitric oxide by the endothelium of internal mammary artery but not of saphenous vein; thromboxane A2 and serotonin mediate contraction in vein but not artery with endothelium. These differences may contribute to differences in graft function and the clinical efficacy of antiplatelet drugs.

Endothelium-dependent regulation of resistance arteries alterations with aging and hypertension

Small arteries with a diameter of 200 μ.m or less play an important role in the regulation of peripheral vascular resistance. Dysregulation of vascular tone of these arteries may contribute significantly to high blood pressure. The contractile state of blood vessels is regulated by peripheral neurons, vascular smooth muscle, and the endothelium. In perfused mesenteric resistance arteries of the rat, removal of the endothelium markedly augments the sensitivity and maximal response to norepinephrine and endothelin-1. Acetylcholine causes profound endothelium-dependent relaxation of resistance arteries contracted with norepinephrine or endothelin-1. The potency of the muscarinic agonist is particularly pronounced with intraluminal application. The inhibitory effects of the endothelium against contractions induced by norepinephrine and endothelin-1 are reduced with aging and hypertension. The endothelium-dependent relaxation in response to intraluminal, but not extraluminal, acetylcholine is blunted in mesenteric resistance arteries of hypertensive rats and in the forearm circulation of hypertensive patients studied in vivo. The sensitivity of vascular smooth muscle to the effects of endothelin decreases with advancing age and in the spontaneously hypertensive rat. Thus, endothelium-derived vasoactive substances can profoundly affect vascular tone of resistance arteries studied in vitro and in vivo. The inhibitory effects of the endothelium against vasoconstrictor stimuli decreases with aging and hypertension, indicating a dysfunction of these regulatory mechanisms under these conditions.