Bioassay Ring Research Articles

Release of endothelium-derived relaxing factor after subarachnoid hemorrhage.

The purpose of this study was to determine the cause of the loss of endothelium-dependent relaxation observed in chronic cerebral vasospasm. A bioassay system was developed to measure the release of endothelium-derived relaxing factor (EDRF) from canine basilar arteries. Subarachnoid hemorrhage (SAH) was induced in dogs by two injections of autologous blood into the cisterna magna. Angiograms were performed on the 7th day after SAH to check the presence of chronic vasospasm. The animals were sacrificed on the 8th day, and in vitro experiments were performed on rings harvested from the basilar artery. These confirmed loss of endothelium-dependent relaxation in response to bradykinin and arginine vasopressin in the group with SAH. The basilar arteries were perfused with modified Krebs-Ringer solution. The perfusate was bioassayed with a ring of coronary artery without endothelium (bioassay ring). The release of the EDRF was detected by relaxation of the bioassay ring contracted with prostaglandin F2 alpha. Arginine vasopressin and bradykinin added to the perfusate upstream of the basilar artery caused concentration-dependent release of the EDRF. The direct effect of these peptides on the smooth muscle of the bioassay ring was to cause contraction. The release of the EDRF was identical in basilar arteries from the control and the SAH groups. These results indicate that the release of the EDRF is not impaired during chronic vasospasm, and thus that the loss of the endothelium-dependent relaxation is due to a decreased transfer of the EDRF or a reduced responsiveness of the smooth muscle to the factor.

Prostacyclin releases endothelium-derived relaxing factor and potentiates its action in coronary arteries of the pig.

1. The possible interactions between prostacyclin and endothelium-derived relaxing factor were examined, in isolated coronary arteries of the pig treated with indomethacin (10(-5) M). 2. In organ chamber experiments, prostacyclin caused relaxations, which were potentiated in the presence of the endothelium; the potentiation was abolished by oxyhaemoglobin. 3. In bioassay experiments, prostacyclin caused minimal relaxations of bioassay rings without endothelium; these relaxations were potentiated when the bioassay ring was exposed to basally-released endothelium-derived relaxing factor (interaction between prostacyclin and basal endothelium-derived relaxing factor) and further augmented when the endothelial cells were exposed to the prostanoid (stimulated release of endothelium-derived relaxing factor). The endothelium-dependent, but not the direct effects of prostacyclin were augmented by superoxide dismutase plus catalase and abolished by oxyhaemoglobin. 4. Forskolin, a direct activator of adenylate cyclase, caused relaxations of rings without endothelium, which were augmented by the presence of the endothelium. 5. The relaxations induced by prostacyclin or forskolin also had an endothelium-dependent component in basilar and femoral arteries and in jugular veins of the pig. 6. The endothelium-dependent actions of prostacyclin probably reflect activation of adenylate cyclase.

Inhibition of adrenergic vasoconstriction by endothelial cell shear stress.

Isolated perfused rabbit carotid arteries were used to determine the effects of endothelial cell shear stress on the response to adrenergic nerve stimulation. Arterial segments with and without endothelium were cannulated and perfused with physiological salt solution. Adrenergic nerves were activated by transmural electrical field stimulation. Neurogenic vasoconstriction was significantly greater in segments without endothelium when compared with that of segments with endothelium. In segments with endothelium only, vasoconstriction was depressed when shear stress was increased by increasing the viscosity of the perfusate with dextran. Perfusion with methylene blue (2 X 10(-6) M), a guanylate cyclase inhibitor, increased vasoconstriction in segments with endothelium only. In the presence of methylene blue, vasoconstriction was no longer different between segments with and without endothelium, and perfusion with dextran had no effect. In a perfusion-cascade system, perfusion with dextran of donor segments with but not without endothelium caused further relaxation of a contracted bioassay ring. These results suggest that shear stress on endothelial cells modulates adrenergic vasoconstriction by augmenting release of endothelial cell-derived vasodilators.

Endothelium-derived relaxing factor release associated with increased endothelial cell inositol trisphosphate and intracellular calcium

The release of eicosanoids and endothelium-derived relaxing factor (EDRF) from endothelial cells is thought to involve a calcium-dependent step. Using cultured bovine aortic endothelial cells as a model system, we have examined the relation between agonist-induced changes in inositol polyphosphates and calcium levels within the endothelial cells and extracellular calcium on EDRF release. In a superfusion-cascade system, EDRF was detected by the relaxation of a rabbit aortic ring without endothelium suspended beneath a column of cultured endothelial cells. Endothelial cell stimulation by bradykinin or melittin induced dose-dependent relaxation of the bioassay ring. In addition, bradykinin and melittin stimulated an increase in intracellular calcium concentration in fura-2 loaded endothelial cells and an increase in inositol 1,4,5-trisphosphate (Ins[1,4,5]P 3) in cells prelabeled with 3H-myoinositol. Bradykinin stimulation produced transient increases in Ins(1,4,5)P 3, fura-2 fluorescence and transient EDRF release. Melittin stimulation induced more prolonged release of EDRF from the endothelial cell column, which was correlated with sustained increases in the fura-2 signal and the level of Ins(1,4,5)P 3. Omission of calcium from the cell superfusate attenuated, but did not eliminate, bradykinin-induced EDRF release and the calcium transient, whereas the melittin-induced responses were only slightly attenuated. Endothelial cells clearly demonstrate receptor-activation of phospholipase C and release of sequestered calcium from subcellular sites in response to Ins(1,4,5)P 3. These results imply that EDRF release is correlated with increased intracellular calcium levels seen in the absence of extracellular calcium. However, sustained release of EDRF does require influx of extracellular calcium via an undefined mechanism.

Endothelium-dependent pressure-induced contraction of isolated canine carotid arteries.

Experiments were conducted in a bioassay system, where a canine coronary artery ring without endothelium (bioassay tissue) was superfused by the effluent from a perfused canine carotid artery segment with endothelium (donor segment). A rapid increase in transmural pressure (from near 0 to 32-38 mmHg) triggered active contraction of the donor segment and simultaneously of the bioassay tissue. These contractions were prevented by removal of the endothelium from the donor segment but not by treatment of the segment with indomethacin. Exposure to elevated pressure depressed basal, acetylcholine-, and flow-induced release of endothelium-derived relaxing factor(s). Methylene blue prevented the pressure-induced contraction of the bioassay ring. These data show that pressure-induced contraction of isolated carotid arteries is endothelium dependent and is mediated by the depression of the synthesis and/or release of endothelium-derived relaxing factor(s).

Potassium-induced release of endothelium-derived relaxing factor from canine femoral arteries.

Experiments were designed in a bioassay system to analyze the effect of elevated (from 5.9 mM to 7.5-45.9 mM) extracellular K+ concentration on the release of endothelium-derived relaxing factor. Segments of canine femoral artery with endothelium (donor segment) were mounted in an organ bath and perfused with modified Krebs-Ringer bicarbonate solution; the effluent from the donor segment was used to superfuse a canine coronary artery ring without endothelium (bioassay tissue). Elevation of perfusate K+ concentration by 1.6-15 mM by intraluminal infusion of potassium chloride upstream of the donor segment evoked further contractions of bioassay rings contracted with prostaglandin F2 alpha. In contrast, the bioassay rings progressively relaxed when increasing concentrations of potassium chloride (10-40 mM) were added extraluminally to the organ bath where the perfused segment was mounted. Extraluminal application of phenylephrine or prostaglandin F2 alpha did not evoke relaxations in the bioassay ring. Removal of the endothelium from the donor segment or selective exposure of the segment (but not the bioassay ring) to Ca2+-deficient solution prevented the K+-induced relaxations. Treatment of the donor segment and the bioassay ring with inhibitors of known endogenous vasoactive substances (acetylcholine, norepinephrine, adenine nucleotides, and prostanoids) had no significant effect on the relaxation of the bioassay ring evoked by extraluminal application of potassium chloride. Simultaneous measurements of changes in isometric force in the donor segment and bioassay ring revealed that extraluminal elevation of K+ concentration relaxed the segments as well and that the relaxations could not be prevented by simultaneous intraluminal infusion of potassium chloride.(ABSTRACT TRUNCATED AT 250 WORDS)

Potassium-free solution prevents the action but not the release of endothelium-derived relaxing factor

Exposure to a K +-free solution reversibly inhibited the acetylcholine-induced relaxation in perfused canine femoral artery segments with endothelium and in superfused bioassay femoral artery rings without endothelium. Infusion of 6.7 mM K + into the K +-free perfusate downstream of the perfused artery restored the acetylcholine-induced relaxation in the bioassay ring, whereas no relaxation was observed in the perfused segment still exposed to the K +-free solution. These data demonstrate that the K +-free solution depresses the endothelium-dependent relaxation to acetylcholine in femoral arteries by preventing the action, but not the production, of endothelium-derived relaxing factor.

Heterogeneity of Endothelium-Dependent Responses to Acetylcholine in Canine Femoral Arteries and Veins

The purpose of this study was to determine whether heterogeneity in endothelium-dependent responses to acetylcholine between canine blood vessels of different anatomical origin reflects variations in endothelial function or in responsiveness of vascular smooth muscle cells. Experiments were conducted in a bioassay system, where segments of femoral artery or vein with endothelium were perfused intraluminally and the perfusate used to superfuse rings of femoral arteries or veins without endothelium. Indomethacin was present in all experiments to prevent the synthesis of prostanoids. The blood vessels were contracted by phenylephrine. Measurement of wall tension in both the perfused segment and bioassay ring allowed simultaneous detection of endothelium-derived relaxing factor(s) released abluminally (segment) and intraluminally (ring). Intraluminal infusion of acetylcholine (ACh) induced relaxations in the perfused artery but not in vein segments. During arterial superfusion ACh induced relaxation in femoral arterial rings but contraction in venous rings. After treatment with atropine the arterial perfusate evoked relaxations in venous rings. Infusion of ACh through the femoral vein evoked only moderate relaxations in arterial rings. These data demonstrate that depressed endothelium-dependent relaxation to ACh in femoral veins compared to femoral arteries is due to a masking effect of the direct stimulating action of ACh and decreased release of the same mediator or the release of a different relaxing factor from venous endothelium.

Nature of endothelium-derived relaxing factor: are there two relaxing mediators?

The role of arachidonic acid in forming endothelium-derived relaxing factor remains controversial. This controversy may be explained if more than one factor exists. To test this hypothesis, the effects of various inhibitors of arachidonic acid metabolism were studied. The perfusate from canine femoral arteries with endothelium was bioassayed with coronary artery rings without endothelium. Treatment of the perfused segment (but not the bioassay ring) with inhibitors of phospholipase A2 (quinacrine) or cytochrome P450 (metyrapone) had no effect on the basal relaxing activity of the effluent; treatment with the inhibitor of lipoxygenase, nordihydroguaiaretic acid, significantly depressed it. With increasing concentrations of acetylcholine, a biphasic concentration-relaxation curve was obtained; the enzyme inhibitors depressed or prevented the first phase but did not affect the second phase. Infusion of arachidonic acid or soybean lipoxidase directly on the bioassay ring did not cause relaxation; together, they evoked concentration-dependent relaxations. These data suggest that acetylcholine can trigger the release of two chemically different relaxing mediators from the endothelium of the canine femoral artery. One factor may be a product of lipoxygenase (or epoxigenase). The second factor is not a metabolite of arachidonic acid and may be released under basal conditions. The existence of two (or more) chemically different endothelium-derived mediators may help to explain the controversial data regarding the nature of the factor(s).

Endothelium inhibits responses of rabbit carotid artery to adrenergic nerve stimulation.

Transmural electrical stimulation of isolated ring segments of the rabbit carotid artery caused frequency-dependent contractions; these were blocked by tetrodotoxin or prazosin. Mechanical or chemical removal of the endothelium markedly augmented responses to electrical stimulation. Inhibition of norepinephrine uptake and metabolism with cocaine, hydrocortisone, and pargyline increased contractions in rings with endothelium more than those without endothelium, but responses remained greater in rings denuded of endothelium. Methylene blue, an inhibitor of guanylate cyclase, enhanced responses to electrical stimulation of rings with intact endothelium only. Combined inhibition of guanylate cyclase and norepinephrine disposition increased the contractions and abolished the difference between the responses of rings with and without endothelium. In a perfusion-cascade system, the perfusate of donor segments with endothelium relaxed a bioassay ring without endothelium. Electrical stimulation of the segment caused no further relaxation of the bioassay ring. However, contractions caused by electrically stimulating the bioassay ring were depressed during superfusion with the perfusate of segments with, but not without, endothelium, indicating that vasodilators spontaneously released from the endothelium inhibit responses to nerve stimulation. These observations suggest that inhibition by the endothelium of the response to adrenergic nerve stimulation results from 1) spontaneous release of endothelium-derived vasodilators and 2) disposition of norepinephrine by the endothelial cells.

Flow-induced release of endothelium-derived relaxing factor.

To analyze the potential mediator(s) involved in flow-induced endothelium-dependent vasodilation, we measured the wall tension of intraluminally perfused canine femoral artery segments and compared the content of 6-ketoprostaglandin F1 alpha (determined by radioimmunoassay) and the relaxing activity of the effluent (determined by bioassay on canine coronary artery rings). During perfusion at a steady flow of 2 ml/min the effluent contained 6-keto-prostaglandin F1 alpha and relaxed the bioassay rings. Sudden increase in steady flow rate to 4 ml/min, or the introduction of pulsatile flow, increased the release of 6-keto-prostaglandin F1 alpha and induced further relaxations of the bioassay ring. No relaxations were observed with the effluent passing through a femoral artery segment without endothelium. Indomethacin significantly depressed the release of 6-keto-prostaglandin F1 alpha during increases in flow but had no significant effect on the relaxing activity of the effluent. In the presence of indomethacin, increases in flow produced significant relaxation in the perfused femoral artery segments with endothelium. Superoxide dismutase restored the relaxing activity of the effluent during increases in flow at a transit time of 30 seconds. These data demonstrate that in addition to prostacyclin, flow triggers the release of another relaxing substance (or substances) from vascular endothelial cells that has characteristics similar to the endothelium-derived relaxing factor released by acetylcholine.

Superoxide anions and hyperoxia inactivate endothelium-derived relaxing factor.

Experiments were designed to determine the effects of oxygen-derived free radicals on the production and biological activity of endothelium-derived relaxing factor or factors released by acetylcholine. Rings of canine coronary arteries without endothelium (bioassay rings) were superfused with solution passing through a canine femoral artery with endothelium. Superoxide dismutase caused maximal relaxation of the bioassay ring when infused upstream, but not downstream, of the femoral artery; this effect of superoxide dismutase was inhibited by catalase. Infusion of acetylcholine relaxed the bioassay rings because it released a labile relaxing factor (or factors) from the endothelium. When infused below the femoral artery, superoxide dismutase and, to a lesser extent, catalase augmented the relaxations to acetylcholine. Superoxide dismutase, but not catalase, doubled the half-life of the endothelium-derived relaxing factor(s). This protective effect of the enzyme was augmented fivefold by lowering the oxygen content of the perfusate from 95 to 10%. These data demonstrate that: superoxide anions inactivate the relaxing factor(s) released by acetylcholine from endothelial cells and hyperoxia favors the inactivation of endothelium-derived relaxing factor(s).