Vanhoutte, Paul M.

Experiments were designed to determine the endothelium-dependent and endothelium-independent responses to aggregating platelets in porcine pulmonary arteries. Isolated rings with and without endothelium from large (5-7-mm-diameter) and small (2-3-mm-diameter) pulmonary arteries were suspended in modified Krebs-Ringer bicarbonate solution bubbled with 95% O2-5% CO2 in the presence of indomethacin. Aggregating platelets caused relaxations in rings with endothelium but contractions in rings without endothelium, both of which were significantly larger in small versus large pulmonary artery rings. Serotonin and ADP caused concentration-dependent endothelium-augmented relaxations that were unaffected by ketanserin. Methiothepin, but not apyrase, significantly decreased the platelet-induced endothelium-dependent relaxations; the residual relaxation was abolished when rings were incubated with methiothepin, apyrase, and theophylline but was unaffected if apyrase was absent, indicating that ADP is responsible for the residual relaxation caused by aggregating platelets. Quiescent rings, with and without endothelium, contracted in a dose-dependent manner to norepinephrine and histamine but not to serotonin or vasopressin. The contraction to aggregating platelets was blocked by methiothepin, pyrilamine, and diphenhydramine but was unaffected by phentolamine, ketanserin, or incubation of the platelets with dazoxiben. These data indicate that, in large and small porcine pulmonary arteries, serotonin and ADP are the major contributors to the endothelium-dependent relaxation caused by aggregating platelets, while histamine appears to be responsible for the contraction that platelets cause in rings without endothelium.

Acetylstrophanthidin, 0.3 to 10 µg/ml, caused contraction of helical strips of the dog cutaneous vein in an organ bath at 37°C. A latent period of 12 to 60 minutes preceded the contraction. During this time, the vein exhibited a progressive increase in response to transmural electric stimulation and added norepinephrine. As the dose of acetylstrophanthidin was increased, the latent period shortened and the increased response during the latent period was potentiated. When the contraction caused by 10 µg/ml was maximal, no additional contraction occurred with electric stimulation; the response to electric stimulation was only partially restored by the repeated washing out of the drug, despite relaxation of the strip to almost base-line tension. Since transmural electric stimulation causes contraction by release of tissue catecholamines, these experiments demonstrate that digitalis glycosides sensitize the cutaneous veins of the dog to adrenergic stimulation. This was confirmed in dogs anesthetized with pentobarbital. Intravenous injections of acetylstrophanthidin and digoxin, in doses too small (12.5 to 15.5 µ/kg) to cause direct constriction of the lateral saphenous vein, potentiated the constriction of this vein caused by electric stimulation of the lumbar sympathetic chain.

Changes in the isometric tension of isolated strips of cutaneous, femoral, mesenteric, pulmonary, and muscle arteries and veins were recorded at 37°C in an organ bath. Acetylcholine (5 x 10 -8 and 10 -7 g/ml) caused relaxation of strips from the saphenous veins, the femoral veins, and all of the arteries after contraction by norepinephrine released from nerve terminals by electrical stimulation (2-5 Hz); in the pulmonary and mesenteric veins, acetylcholine caused a further increase in tension. Pulmonary artery and mesenteric vein strips were incubated with [ 3 H] norepinephrine and mounted for superfusion (3 ml/min) and isometric tension recording. Electrical stimulation increased the tension and the total radioactivity released in both preparations. Acetylcholine (2 x 10 -7 g/ml) depressed the contractions of the pulmonary artery strips but augmented those of the mesenteric vein strips; it diminished the efflux of radioactivity in both, indicating that acetylcholine inhibits adrenergic neurotransmission. In the absence of sympathetic stimulation, acetylcholine (5 x 10 -10 -10 -5 g/ml) caused all vein strips to contract; the most common reaction in artery strips was a slight relaxation (at 10 -9 -10 -8 g/ml) followed by a contraction (at 5 x 10 -8 -10 -5 g/ml). During contractions caused by norepinephrine, acetylcholine caused a further increase in tension in vein strips but a relaxation in artery strips. Atropine abolished the effects of acetylcholine. The results of this study suggest the presence in vascular smooth muscle of both excitatory and inhibitory cholinergic receptors.

The present study examined the protective role of the venous endothelium against aggregating platelets and its modulation by diet. Yorkshire pigs were fed a regular chow (control pigs), 2% high-cholesterol diet (for 10 weeks, cholesterol-fed pigs), and regular chow plus cod-liver oil (30 ml/day for 4 weeks, oil-fed pigs). Endothelium-dependent responses were examined in vitro in rings of femoral veins in the presence of the inhibitor of cyclooxygenase indomethacin. In control pigs, aggregating platelets, serotonin, and ADP caused endothelium-dependent relaxations. The platelet-induced relaxations were attenuated by methiothepin (a combined 5-HT1 and 5-HT2 serotonergic blocker) or apyrase (an ADPase and ATPase) and were abolished by the combination of the two agents. In quiescent rings, platelets caused contractions, which were reduced in the presence of endothelium; the contractions were prevented by ketanserin (a 5-HT2 serotonergic blocker) or methiothepin but not by R 68 070 (a thromboxane A2 receptor blocker) or dazoxiben (a thromboxane-synthetase blocker). In cholesterol-fed pigs, the platelet-induced relaxations were not altered, whereas in oil-fed pigs, the endothelium-dependent relaxations to platelets, serotonin, and ADP were augmented. Platelet-induced contractions were significantly reduced in rings with endothelium from oil-fed pigs, whereas the contractions were comparable in rings without endothelium among the three groups. Endothelium-dependent relaxations in response to the calcium ionophore A23187, direct relaxations in response to sodium nitroprusside, and direct contractions in response to potassium chloride were comparable among the three groups.(ABSTRACT TRUNCATED AT 250 WORDS)

To study the effect of dietary supplementation with fish oil on endothelium-dependent responses, Yorkshire pigs were maintained on a normal diet or on a low (0.6 ml/kg/day) or a high (1.0 ml/kg/day) dose of cod-liver oil for 4 weeks. Endothelium-dependent responses were examined in vitro in rings of proximal left anterior descending coronary arteries taken from control and treated animals studied in parallel. Endothelium-dependent relaxations in response to bradykinin, serotonin, adenosine diphosphate, and thrombin were facilitated in arteries from treated but not in those from control animals, whereas the relaxations in response to A23187 were unaltered. The facilitated relaxations were not altered by indomethacin but significantly inhibited by methylene blue. Aggregating platelets from control and treated pigs induced comparable, facilitated endothelium-dependent relaxations in rings taken from treated pigs. The platelet-induced contractions were significantly reduced in rings with endothelium taken from treated pigs, and they were comparable in rings without endothelium in both groups. Aggregating platelets from control and treated pigs released comparable amounts of serotonin and thromboxane A2. Endothelium-dependent relaxations induced by arachidonic acid and eicosapentaenoic acid were unaltered, whereas transient endothelium-dependent contractions induced by arachidonic acid were significantly reduced by the treatment with cod-liver oil. Relaxations to sodium nitroprusside or isoproterenol,and contractions to potassium chloride or serotonin were not different in rings without endothelium from control or treated pigs. These results indicate that dietary supplementation with cod-liver oil facilitates endothelium-dependent relaxations and inhibits endothelium-dependent contractions in porcine coronary arteries.

The effects of the calcium ionophore A23187, arachidonic acid, and acetylcholine were studied in isolated canine basilar arteries. Rings with and without endothelium were suspended for isometric tension recording in physiological saline. In unstimulated rings, A23187, arachidonic acid, and acetylcholine caused endothelium-dependent contractions. The contractions of rings caused by uridine 5'-triphosphate were not affected by removal of the endothelium. An inhibitor of cyclooxygenase, indomethacin (10(-5) M), prevented excitatory responses to A23187, arachidonic acid, and acetylcholine but did not alter contractions caused by KCl. An inhibitor of thromboxane synthetase, dazoxiben (10(-4) M), significantly reduced endothelium-dependent contractions to A23187 and arachidonic acid but did not significantly affect contractions caused by acetylcholine. These results demonstrate that A23187, arachidonic acid, and acetylcholine cause excitatory endothelium-dependent responses in canine cerebral blood vessels by increasing the release of product(s) of cyclooxygenase from endothelial cells; in the case of A23187 and arachidonic acid, thromboxane A2 contributes to the endothelium-dependent contractions.