T.M. Cocks

Vasodilatation by acetylcholine is endothelium‐dependent: a study by sonomicrometry in canine femoral artery in vivo.

External diameter of the femoral artery was measured by sonomicrometry in the anaesthetized dog. Intra‐arterial acetylcholine lowered arterial pressure and thereby passively lowered diameter. When blood flow and distal resistance were controlled by roller pump and Starling resistor respectively, acetylcholine (0.1‐10 microM) and substance P (0.1‐1 nM) both caused up to 10% increase in diameter. Removal of endothelium by mechanically rubbing the artery lumen abolished the dilator response to acetylcholine and substance P but did not affect the response to nitroprusside. Constrictor responses to noradrenaline were unaltered by endothelium removal. Topical application of acetylcholine and substance P onto the adventitial surface of the artery also caused an increase in diameter but both agents were 50‐100 times less potent by this route compared with intra‐arterial infusion. These dilator responses were abolished by endothelium removal. In these circumstances acetylcholine caused constriction. We conclude that acetylcholine and substance P require an intact endothelium to elicit vasodilatation in vivo, at least for the large femoral artery. The results from the topical application experiments suggest that local neural release of vasoactive substances such as acetylcholine and substance P depend on an intact endothelium to cause vasodilatation.

ENDOTHELIN IS BLOOD VESSEL SELECTIVE: STUDIES ON A VARIETY OF HUMAN AND DOG VESSELS IN VITRO AND ON REGIONAL BLOOD FLOW IN THE CONSCIOUS RABBIT

1. Endothelin (Et), a vasoconstrictor peptide, was 5–10‐fold more potent (lower EC50) on isolated ring segments of large veins than on large arteries removed from dog coronary, mesenteric, femoral, renal and internal mammary vasculature and from the human internal mammary pedicle.

THE ACETYLCHOLINE PARADOX: A CONSTRICTOR OF HUMAN SMALL CORONARY ARTERIES EVEN IN THE PRESENCE OF ENDOTHELIUM

1. In animal experiments, acetylcholine is generally a vasodilator acting indirectly by releasing endothelium‐derived relaxing factor (EDRF); for example, in dog and rabbit small coronaries mounted in a myograph, acetylcholine caused concentration‐dependent relaxation.

The half-life of endothelium-derived relaxing factor released from bovine aortic endothelial cells in culture.

1. The half‐life of endothelium‐derived relaxing factor (EDRF) in Krebs solution was determined by bioassay in vitro. 2. A column of bovine aortic endothelial cells grown on microcarrier beads in suspension culture was perfused with Krebs solution. EDRF was released from these cells by sequential treatment with increasing concentrations of bradykinin (0.01‐100 nM). EDRF was detected by the relaxation of an endothelium‐denuded ring segment of dog coronary artery. 3. Complete bradykinin concentration‐relaxation curves were determined in the absence or presence of coils of tubing that increased the transit time (delay) between the cell column and the assay tissue. An estimate of the falls in concentration, and hence of the half‐life of EDRF, was obtained from the shift of the bradykinin concentration‐relaxation curves. 4. Mass‐action equations were used to model the relationship between the indirectly acting agonist bradykinin and the relaxation via EDRF. The modelling adequately predicted the consequences of different transit delay times (0‐4 half‐lives). 5. This new analysis of half‐life of an active intermediate emphasizes the measurement of changes in concentration with increasing transit time rather than a fall in tissue response. 6. The half‐life of EDRF in Krebs solution is 41 s.

Evidence that contractions of isolated arteries by L-NMMA and NOLA are not due to inhibition of basal EDRF release

We have tested the hypothesis the endothelium-dependent contractions to two L-arginine analogues modified in either of the guanidino nitrogens [e.g., N G -monomethyl-L-arginine (L-NMMA) and N w -nitro-L-arginine (NOLA)] are due to inhibition of basally released EDRF. We found that these contractions in the dog isolated coronary artery were maximal at 10 μM for each compound, as increasing the concentration 10-fold further gave no significant increase in developed force.

Development of a Large Fibromuscular Intimal Thickening Does Not Impair Endothelium-Dependent Relaxation in the Rabbit Carotid Artery

The release of endothelium-derived relaxing factor (EDRF) was examined in the rabbit carotid artery 6 weeks after denudation with an inflated balloon catheter in vivo. A concentric, fibromuscular intimal thickening of variable width developed in all areas lined with either regenerated endothelium or modified luminal smooth muscle cells. In vitro studies showed that in vessels precontracted with serotonin, only the re-endothelialized areas could relax to the endothelium-dependent dilators methacholine, substance P and the Ca2+ ionophore A23187. Re-endothelialized areas with large concentric, fibromuscular intimal thickening (between 10 and 20 cells thick) relaxed with a similar sensitivity and maximum to methacholine compared with control areas. It is concluded that newly generated endothelial cells release EDRF whilst the specialized lining smooth muscle cells present 6 weeks after injury do not, and that the presence of a large fibromuscular intima does not prevent EDRF from reaching the media to cause relaxation.

ENDOTHELIUM‐DEPENDENT RELAXATION IS UNALTERED BY HYPERTENSION, CHOLESTEROL OR INTIMAL THICKENING

1. The effect of cellophane wrap hypertension (WRAP) and hypercholesterolemia (1% cholesterol diet, CHOL) for 4 weeks was assessed on the endothelium‐dependent vasodilator response to acetylcholine in conscious rabbits after pharmacological autonomic blockade.

Alpha2-adrenoceptors and endothelium dependent relaxation in canine large arteries

Ring preparations from the carotid, coronary, renal, mesenteric and femoral arteries of the dog were precontracted with the thromboxane mimetic U46619, after ensuring that the resting conditions were comparable from the Laplace relationship. In the presence of prazosin (1 microM) and propranolol (3 microM), noradrenaline (NA) relaxed the arteries in the order coronary greater than carotid greater than femoral greater than renal = mesenteric. When maximum relaxation to nitroglycerin (10 microM) was taken to be 100% the maximum relaxation to noradrenaline in each artery was: coronary 70%; carotid 34%; femoral 19%; renal 7% and mesenteric 2%. In endothelium-intact arteries UK14304 mimicked the relaxation responses to NA and idazoxan shifted the curves to both agonists to the right, consistent with an alpha 2-adrenoceptor classification. Substance P relaxed the arteries in the same order as for NA but showed higher efficacy i.e.: coronary 100%; carotid 80%; femoral 71% renal 49%; and mesenteric 41%. Removal of the endothelium abolished the relaxation to NA. We conclude that endothelium-dependent relaxation to NA and substance P varies greatly across 5 large arteries of the dog. This may indicate that endothelium-derived relaxing factor (EDRF) release is site-dependent or that the efficacy of EDRF on smooth muscle varies; being greatest in the coronary and weakest in the renal and mesenteric arteries.