Bioassay Ring Research Articles

Estrogen modulation of endothelium-derived relaxing factors by human endothelial cells

We report the modulatory effects of estrogen on release of endothelium-derived relaxing factors (EDRFs) in a human endothelial cell line, EA.hy926. Using bioassay, we showed that EA.hy926 released EDRF including nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) measured by relaxation of pre-contracted endothelium-denuded rabbit aortic rings. This EDRF production was significantly higher in cells treated for 24h with 17-β-estradiol (10−6mol/L) than control cells. Addition of l-NAME to the perfusate of cells caused the relaxation induced by the endothelial cell perfusate to become transient and abolished the enhancement of relaxation due to estrogen treatment. Addition of KCa channel blockers to the perfusate abolished the l-NAME-resistant relaxation of the bioassay ring. Using real-time PCR, we demonstrated that eNOS expression in estrogen-treated cells was significantly higher than controls. These results show that estrogen exerts a potentially important vasculo-protective effect by stimulating NO but not EDHF production.

Endothelial sodding of the permaflow prosthetic coronary artery bypass conduit

Background. Experiments were designed to determine the feasibility of sodding an endothelial monolayer within the lumen of a prosthetic conduit applied to the canine coronary circulation. Methods. Autologous endothelial cells were sodded onto the luminal surface of the Permaflow conduit and immediately implanted to bypass the left circumflex coronary artery in adult mongrel dogs (n = 9). Unsodded Permaflow conduits were implanted as controls (n = 8). At 3 weeks, grafts were explanted and examined by scanning electron microscopy and immunostained for canine von Willebrand factor. Results. Sodded grafts contained a confluent endothelial cell layer devoid of adherent thrombus or platelets and stained positively for canine von Willebrand factor. Unsodded grafts contained no endothelium and retained adherent platelets, collagen, and fibrin. Effluent from sodded grafts stimulated with calcium ionophore A23187 caused a significantly greater relaxation of its bioassay ring than effluent from unsodded grafts (60% ± 21% versus 12% ± 5%; n = 8, p < 0.03). Conclusions. Sodding of endothelial cells onto a Permaflow coronary artery bypass graft results in a confluent, viable, nonthrombogenic, endothelial monolayer and releases vasodilator substances in response to calcium ionophore A23187. Endothelial sodding may optimize prosthetic grafts.

Macrophage-induced nitric oxide and prostanoid dependent relaxation of arterial smooth muscles

We have studied mechanisms of vasodilation induced by supernatant fluid of rat macrophages (Mø), using an arterial bioassay preparation. The cells emigrated by an intraperitoneal injection of thioglycollate were isolated and cultured for 12 h in RPMI 1640 medium with and without 2.0 mM L-arginine. More than 98% of the isolated cells clearly demonstrated Wright's esterase staining and phagocytosis of acetylated low-density lipoprotein. The bioassay preparation was made of dog isolated femoral arteries with and without the endothelium. The supernatant of macrophages cultured in the L-arginine-free RPMI 1640 caused a significant reduction of the precontraction in the bioassay rings, being approximately 51.6-66.7% of sodium nitroprusside (SNP) induced maximum vasodilation in each ring. The supernatant of macrophages cultured in the RPMI 1640 containing 2.0 mM L-arginine produced a significantly smaller relaxation (approximately 32.3-33.3%). The Mø-induced vasodilation was significantly inhibited by the coculture of the macrophages with 1 microM dexamethasone, 10 microM cycloheximide, 50 microM N omega-nitro-L-arginine methyl ester (L-NAME), 10 microM indomethacin, or 10 microM aspirin. The L-NAME-induced inhibition was significantly reversed by an additional treatment with 100 microM L-arginine. The coculture with both L-NAME and indomethacin caused a reduction of the Mø-induced vasodilation (approximately 12.5-13.4%) similar to reductions produced by dexamethasone (approximately 10.8-12.1%) and cycloheximide (approximately 11.4-12.4%). Coculture with 10 micrograms/mL bacterial lipopolysaccharide caused a slight facilitation of the Mø-induced vasodilation (approximately 78.2-79.6%). These findings suggest that supernatant fluid of rat exuded macrophages cultured with low concentrations of L-arginine causes an endogenous nitric oxide (NO) and vasodilative prostaglandin dependent relaxation of arterial smooth muscles.

Macrophage-induced nitric oxide and prostanoid dependent relaxation of arterial smooth muscles

We have studied mechanisms of vasodilation induced by supernatant fluid of rat macrophages (Mø), using an arterial bioassay preparation. The cells emigrated by an intraperitoneal injection of thioglycollate were isolated and cultured for 12 h in RPMI 1640 medium with and without 2.0 mM L-arginine. More than 98% of the isolated cells clearly demonstrated Wright's esterase staining and phagocytosis of acetylated low-density lipoprotein. The bioassay preparation was made of dog isolated femoral arteries with and without the endothelium. The supernatant of macrophages cultured in the L-arginine-free RPMI 1640 caused a significant reduction of the precontraction in the bioassay rings, being approximately 51.6-66.7% of sodium nitroprusside (SNP) induced maximum vasodilation in each ring. The supernatant of macrophages cultured in the RPMI 1640 containing 2.0 mM L-arginine produced a significantly smaller relaxation (approximately 32.3-33.3%). The Mø-induced vasodilation was significantly inhibited by the coculture of the macrophages with 1 microM dexamethasone, 10 microM cycloheximide, 50 microM N omega-nitro-L-arginine methyl ester (L-NAME), 10 microM indomethacin, or 10 microM aspirin. The L-NAME-induced inhibition was significantly reversed by an additional treatment with 100 microM L-arginine. The coculture with both L-NAME and indomethacin caused a reduction of the Mø-induced vasodilation (approximately 12.5-13.4%) similar to reductions produced by dexamethasone (approximately 10.8-12.1%) and cycloheximide (approximately 11.4-12.4%). Coculture with 10 micrograms/mL bacterial lipopolysaccharide caused a slight facilitation of the Mø-induced vasodilation (approximately 78.2-79.6%). These findings suggest that supernatant fluid of rat exuded macrophages cultured with low concentrations of L-arginine causes an endogenous nitric oxide (NO) and vasodilative prostaglandin dependent relaxation of arterial smooth muscles.

Activated macrophage-mediated endogenous prostaglandin and nitric oxide-dependent relaxation of lymphatic smooth muscles.

The effects of macrophages activated by bacterial lipopolysaccharides (LPS) on the mechanical activity of lymph vessels with or without the endothelium were investigated using conventional bioassay preparations. Rat peritoneal macrophages emigrated by an injection of thioglycollate were isolated and cultured for 12 h in RPMI 1,640 medium containing 10 micrograms/ml LPS. More than 97% of the cultured cells were stained with monoclonal antibody ED1 and demonstrated phagocytosis of acetylated low-density lipoprotein. The supernatant of the macrophages (M phi) suppressed significantly the basal tone of the lymphatic bioassay rings precontracted by 10(-8) M U46619. The M phi-induced vasodilation of the lymph nodes was significantly reduced by 12 h preincubation of the macrophages with 5 x 10(-5) M N omega-nitro-L-arginine methyl ester (L-NAME), 10(-5) M indomethacin, 10(-6) M dexamethasone, or 10(-5) M cycloheximide. Simultaneous preincubation of L-NAME and indomethacin caused a synergistic reduction of the M phi-induced vasodilation of the lymphatic bioassay rings. The superfusion of Krebs-bicarbonate solution containing 5 x 10(-5) M L-NAME, 5 x 10(-5) M aspirin, or the culture medium with no macrophages caused no significant effect on the M phi-induced vasodilation. These findings suggest that macrophages activated by bacterial LPS produce a marked relaxation of lymphatic smooth muscles through the co-release of nitric oxide and vasodilative prostaglandins, which may result in the facilitation of edema formation in wound tissues.

Production of endothelium-derived factors from sodded expanded polytetrafluoroethylene grafts.

Experiments were designed to determine whether endothelium isolated from adipose tissue and sodded onto expanded polytetrafluoroethylene grafts release endothelium-derived vasoactive factors. Thin-walled expanded polytetrafluoroethylene grafts (6 mm internal diameter, 6 cm length, 30 microm pore size), one sodded with autogenous endothelial cells, the other unsodded, were implanted bilaterally in carotid arteries of 30 male mongrel dogs. Dogs were treated with 325 mg aspirin daily. After 6 weeks grafts were excised and perfused in a bioassay system. Effluent from the grafts stimulated with either acetylcholine, thrombin, adenosine 5-diphosphate, or the calcium ionophore A23187 was superfused over rings of canine femoral arteries without endothelium contracted with phenylephrine. Effluent from the grafts was analyzed by radioimmunoassay for thromboxane B2, 6-keto-prostaglandin F1alpha, endothelin-1, and C-type natriuretic peptide. Ninety percent of the sodded grafts and 87% of the unsodded grafts were patent after 6 weeks. Bioassay rings superfused with effluent from sodded grafts stimulated with acetylcholine relaxed significantly more than rings superfused with effluent from similarly stimulated unsodded grafts. Biochemical analysis of the effluent showed an increase in 6-keto prostaglandin F1alpha and C-type natriuretic peptide and a decrease in endothelin-1 and thromboxane B2 release from the sodded compared with the unsodded grafts. Scanning electron microscopy showed a continuous layer of endothelial cells lining only the sodded grafts. Staining for alpha-actin and heavy-chain myosin showed a differentiated layer of smooth muscle below the endothelial layer on the sodded grafts. Finally, there was positive staining for C-type natriuretic peptide and endothelin-1 in the endothelium of the sodded grafts. These results indicate that endothelial cells of sodded expanded polytetrafluoroethylene grafts produce endothelium-derived vasoactive factors. In addition, receptor-coupled synthesis/release of these factors is retained in sodded endothelial cells.

Acetylcholine- and flow-induced production and release of nitric oxide in arterial and venous endothelial cells.

To study flow-mediated responses in a conduit vein, we investigated the physiological characteristics of endothelium-dependent acetylcholine (ACh)- and flow-induced relaxations using a conventional bioassay cascade. Cylindrical segments isolated from canine common carotid arteries and external jugular veins were perfused at a constant mean flow rate ranging from 1 to 8 ml/min. Endothelium-derived nitric oxide (NO) activity in perfusion effluent through the arterial and venous segments was measured by relaxation of endothelium-denuded arterial rings and arterial and/or venous rings precontracted by prostaglandin F2 alpha, respectively. Stimulation by a flow rate of 8 ml/min on the arterial and venous endothelial cells produced approximately 60 and 20% of the maximum relaxation in the arterial and venous rings, respectively. ACh (10(-6) and 10(-5) M) perfused through the arterial and venous segments with endothelium caused dose-related relaxations of both bioassay rings. The ACh- and flow-induced relaxations were completely reduced by mechanical removal of the endothelial cells. Pretreatment with 5 x 10(-5) M NG-nitro-L-arginine methyl ester (L-NAME) produced a significant reduction of the ACh- and flow-induced vasodilation. Additional treatment with 10(-4) M L-arginine significantly reversed the L-NAME-induced inhibition of ACh-induced relaxation but had no effect on flow-induced relaxation. When the flow rate was increased from 2 to 4 ml/min, the same concentrations of ACh produced larger dose-related relaxations than those obtained at a flow rate of 2 ml/min. Pretreatment with 25 U/ml superoxide dismutase caused no significant effect on the flow-mediated potentiation of ACh-induced relaxation. These findings suggest that venous endothelial cells of canine large vein are able to produce and release NO by stimulation of increased flow or ACh to a significantly lesser extent compared with the artery and that ACh-induced vasodilation is potentiated by an increase in shear stress up to approximately 4 dyn/cm2 loaded on the endothelial cells.

TEA inhibits ACh-induced EDRF release: endothelial Ca(2+)-dependent K+ channels contribute to vascular tone

The effects of K(+)-channel blockers on the acetylcholine (ACh)-induced relaxation of vascular smooth muscle, intracellular free Ca2+ concentration ([Ca2+]i) elevation, and ACh-evoked outward K+ current of endothelial cells of rabbit aorta were studied using bioassay, spectrofluorimetry, and patch-clamp techniques, respectively. In bioassay experiments, ACh caused relaxation of endothelium-denuded aortic rings in a concentration-dependent manner when perfused through an endothelium-intact donor segment of aorta but not when perfused directly onto the recipient aortic ring. ACh-induced relaxation was inhibited by perfusion of tetraethylammonium ions (TEA; 5 mM) through the donor but not by perfusion directly onto the recipient segment. Glibenclamide had no effect on ACh-induced relaxation of the bioassay ring in either situation. ACh increased [Ca2+]i at the endothelial surface of aortic strips but not at the adventitial surface. TEA inhibited ACh-induced [Ca2+]i elevation, whereas glibenclamide had no effect. In patch-clamp experiments with freshly isolated endothelial cells, ACh evoked a biphasic outward current which was completely abolished by TEA (3 mM). It is concluded that Ca(2+)-dependent K+ channels are important for increasing [Ca2+]i during agonist stimulation and consequently for the synthesis/release of endothelium-derived relaxing factors (EDRFs). Furthermore, endothelial ATP-sensitive K+ channels do not contribute to ACh-induced relaxation or evoke an increase in endothelial [Ca2+]i of rabbit thoracic aorta.

Chronic treatment with the anabolic steroid, nandrolone, inhibits vasodilator responses in rabbit aorta

The effect of chronic treatment of rabbits for 4, 8 and 12 weeks with the anabolic steroid, nandrolone, on vasodilator responses was studied in segments of different arteries. The treatment abolished endothelium-dependent relaxation caused by acetylcholine and the Ca 2+-ionophore, A23187, in thoracic aorta, and reduced endothelium-independent relaxations induced by exogenous nitric oxide (NO) or sodium nitroprusside. The inhibitor of NO synthesis, N G-monomethyl- l-arginine, abolished vasodilator responses to acetylcholine and A23187. In contrast, relaxation induced by acetylcholine, NO or sodium nitroprusside in mesenteric and femoral arteries was unaltered by nandrolone treatment. Bioassay experiments using donor segments and endothelium-denuded bioassay rings from thoracic aorta show that acetylcholine, applied either through control or treated (12 weeks) donor segments, produced similar relaxation in bioassay rings from control rabbits, but this relaxation was markedly reduced in rings from treated rabbits. The increases of cyclic GMP levels induced by acetylcholine or sodium nitroprusside in segments from thoracic aorta were abolished by nandrolone treatment. These results suggest that the treatment with nandrolene reduces NO-mediated relaxation only in thoracic aorta by inhibition of guanylate cyclase, endothelial NO production and vasodilator machinery being unaltered.

Adenylate cyclase‐mediated vascular responses of rabbit aorta, mesenteric artery and skin microcirculation

1. The importance of adenylate cyclase-mediated vascular relaxation in the macro and microcirculation was assessed in rabbit aortic and coeliac artery bioassay rings in vitro and skin microvessels in vivo. 2. The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP38), the beta-agonist, isoprenaline, and the prostaglandins, PGE1 and PGE2, were compared with the activity of nitroprusside, which acts by stimulating guanylate cyclase. 3. In aortic tissue the relative relaxant potencies were (-log M EC50, 100% = response to nitroprusside 10(-6) M): nitroprusside 7.0, PACAP38 6.8, isoprenaline 6.3; PGE1 and PGE2 were weak constrictors. In coeliac artery rings relative potencies were (-log M EC50, 100% = response to nitroprusside 10(-5) M): PACAP38 6.6, PGE1 6.6, nitroprusside 6.5, PGE2 4.9, and isoprenaline 4.3. 4. Comparative potencies when injected into anaesthetized rabbit skin in vivo were (-log mol/site required to increase blood red cell flux by 75%): PACAP38 13.0, PGE2 10.7, isoprenaline 9.7, PGE1 9.1, nitroprusside < 7. 5. Nitroprusside, the most effective relaxant tested in the aorta, was 10(7) fold less potent than PACAP in its effect on skin blood flow. PGE1 and PGE2 were constrictors of the aorta, of intermediate effect in the coeliac artery, but potent vasodilators of the microcirculation. 6. In this model, the importance of adenylate cyclase-mediated vascular relaxation increases with decreasing vessel size.

Bioassay of EDRF from internal mammary arteries: Implications for early and late bypass graft patency

To study the basal, luminal release of endothelium-derived relaxing factor, 35-mm segments of canine internal mammary artery (IMA) were cannulated and perfused at 5 mL/min in vitro with physiological salt solution. Vasoactive properties of the effluent were bioassayed on coronary artery smooth muscle. Effluent from IMAs produced significant vasodilation of the bioassay ring compared with effluent from a prosthetic conduit (n = 24; p < 0.05). The vasodilation by the effluent could be eliminated by mechanically removing the intima of the IMA, or by treating the IMA segments with N G -monomethyl- l-arginine or N G -nitro- l-arginine, two competitive inhibitors of nitric oxide synthesis from l-arginine; vasodilation was not influenced by treatment with indomethacin. In 83% of the superfusion experiments, effluent from the left IMA induced greater relaxation of the bioassay ring than did effluent from the right IMA. In addition, the average vasodilation induced by left IMA effluent was 28% ± 2.3% versus 17.4% ± 3.1% for the right (n = 24; p < 0.05). However, in organ chamber experiments, right and left IMAs exhibited comparable endothelium-dependent vasodilation to acetylcholine (n = 6). Because endothelium-derived relaxing factor induces vasodilation and also inhibits platelet adhesion, platelet aggregation, and atherogenesis, luminal release of endothelium-derived relaxing factor by the IMA could contribute to superior results when the artery is used in bypass grafting.

Bioassay of endothelium-derived relaxing factor in diabetic rat aorta.

The bioassay technique was utilized to quantitate endothelium-derived relaxing factor (EDRF) released from perfused donor segments of control and diabetic rat aorta. In the presence of indomethacin, perfusates of donor segments with endothelium were allowed to superfuse recipient detector rings of normal rat aorta without endothelium. Under basal conditions, relaxations of the bioassay rings to perfusates of control and diabetic donor segments were similar. Perfusion of donor segments with acetylcholine produced relaxation of bioassay rings, which was decreased from endothelial perfusion of diabetic donor segments. These relaxations were inhibited by addition of methylene blue to the detector ring or by perfusion of donor segments with nitro-L-arginine. Infusion of superoxide dismutase (SOD) at a site proximal to the donor segment normalized relaxations induced by acetylcholine addition to diabetic donors. In contrast, infusion of SOD distal to the donor had no effect on acetylcholine-stimulated relaxations of detector rings from control donors while attenuating, paradoxically, the relaxations of detector rings from diabetic donors. These results suggest that diabetic rat aortas release similar levels of EDRF in response to acetylcholine, but the action of EDRF arising from diabetic donors is attenuated by enhanced release of oxygen-derived free radicals, which limits EDRF-mediated relaxation of vascular smooth muscle.

Humoral factor mediates acetylcholine-induced endothelium-dependent relaxation of chicken aorta

A superfusion cascade system was used to determine whether endothelium-dependent vasorelaxation induced by acetylcholine (ACh) is mediated by a humoral factor(s) in the domestic fowl. An abdominal aorta (5 cm in length) with intact endothelium, excised from the donor bird, was mounted in an organ bath and perfused with avian Ringer solution. The vasorelaxing activity of the effluent was determined by an endothelium-denuded aortic ring (bioassay ring) equilibrated and precontracted with phenylephrine (PHE) (10 −6 M). The effluent (containing PHE) from the donor aorta with intact endothelium did not significantly change the PHE-induced tension of the bioassay rings. ACh (10 −6 M), when added to the perfusion medium that runs through the control polyethylene tubing, produced further contraction of the PHE-precontracted endothelium-denuded bioassay rings, whereas ACh caused relaxation of the rings when added to the perfusate that passes through the endothelium-intact donor aorta. The ACh-induced relaxation of the bioassay ring was sightly potentiated by superoxide dismutase (100 U/ml). Hemoglobin (10 −5 M) increased the basal tension of the bioassay rings and completely inhibited the ACh-induced relaxation of the rings. These results suggest that ACh-induced relaxation of fowl aorta is, at least partially, mediated by a humoral factor(s) that resembles endothelium-derived relaxing factor demonstrated in mammals.

Phorbol dibutyrate stimulates the release of diffusible endothelium-derived vasoconstrictor factor(s) from canine femoral arteries.

The purpose of this study was to analyze the effect of the tumor-promoting phorbol ester 12,13-dibutyrate (PDBu) on the synthesis/release of nonprostanoid endothelium-derived vasoactive factors. In bioassay experiments (in the presence of 10(-5) M indomethacin), infusion of PDBu (10(-9)-10(-7) M) through a femoral artery (donor) segment with endothelium evoked further, concentration-dependent contraction of superfused canine coronary artery bioassay rings without endothelium (already contracted with 10(-7) M PDBu). Removal of the endothelium from the donor segment abolished further contractions of the bioassay ring to 10(-9) M PDBu and significantly depressed the contractile responses to 10(-9) and 10(-7) M PDBu infused through the donor segment. The inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate had no effect on vascular preparations mounted in the bioassay system. Selective exposure of the bioassay tissue to 10(-7) M PDBu completely inhibited its responsiveness to basally released endothelium-derived relaxing factor. These data indicate that PDBu stimulates the release of a diffusible and bioassayable vasoconstrictor mediator(s) from the endothelium of canine femoral arteries.

Endothelium-derived relaxing and contracting factors: Perspectives in nephrology

The fact that the endothelium can profoundly affect the function of vascular smooth muscle cells has been recognized only recently. In 1980, Furchgott and Zawadzki demonstrated that the relaxation of isolated arteries induced by acetylcholine requires the presence of endothelial cells (Fig. 1) [1]. Bilayer experiments in which the relaxation induced by the muscarinic agonist could be transferred from a vascular segment with endothelium, to one without, indicated that an endothelium-derived diffusible substance must be involved [1–3]. This was confirmed in bioassay experiments demonstrating that arterial rings without endothelium (bioassay ring) relax in response to acetylcholine or bradykinin only if they were supervised with physiological salt solution passing through either a column with cultured endothelial cells or an arterial segment with endothelium [4–7]. The unknown substance was named endothelium-derived relaxing factor (EDRF) [1–8] (Fig. 2). More recently it has been recognized that the endothelium can also produce constrictor substances [2, 3]. This article updates previous reviews on endothelium-derived relaxing and contracting factors [9–11] and focuses on their potential importance in nephrology.

Release of EDRF from canine renal artery by leukotriene D4

The goal of the present investigation was to determine whether leukotriene D4 (LTD4) was capable of releasing endothelium-derived relaxing factor (EDRF) from perfused renal arterial (RA) segments and to begin to characterize any released mediator. To detect EDRF, a bioassay was used in which a prostaglandin (PG) F2 alpha precontracted, endothelium-denuded coronary artery (CA) ring was superfused either directly or with effluent from a perfused RA segment. Addition of LTD4 or acetylcholine (ACh) to the perfusate proximal to the RA segment resulted in CA ring relaxation, the degree of which was inversely related to transit time. Calculated half-life (t1/2) for the CA relaxing substance released by LTD4 and ACh from the RA segment was 7.5 +/- 1.4 and 7.4 +/- 0.9 s, respectively. Pretreatment of the RA segment with collagenase prevented relaxation of the CA ring evoked by RA effluent in response to either ACh or LTD4 that was administered into the perfusate proximal to the RA segment. Addition of superoxide dismutase (SOD) to the effluent distal to the RA segment markedly enhanced both ACh and LTD4-evoked relaxation of the CA ring, whereas reduced hemoglobin (Hb) virtually abolished these responses. When either LTD4 or ACh was superfused directly over the CA, no change in tone of the bioassay ring was observed. These data indicate that, similar to ACh, LTD4 has the ability to release a labile substance(s), presumably EDRF, from the endothelium-intact RA segment that evokes relaxation of the endothelium-denuded CA ring. Although apparently similar, further studies are required to confirm whether or not the mediator(s) released by LTD4 is identical to that which is released by ACh.

Chronic exposure of cultured endothelial cells to eicosapentaenoic acid potentiates the release of endothelium‐derived relaxing factor(s)

1. The effect of chronic exposure of cultured porcine aortic endothelial cells to eicosapentaenoic acid on the release of indomethacin-insensitive relaxing factor(s) was investigated (a) under bioassay conditions using preconstricted canine coronary artery rings without endothelium and (b) by the measurement of guanosine 3':5'-cyclic monophosphate (cyclic GMP) content of endothelial cells. 2. Exposure of endothelial cells for 8-10 days to eicosapentaenoic acid (2.5 x 10(-5) M) did not affect the relaxing activity of the perfusate from unstimulated endothelial cells. 3. The treatment with eicosapentaenoic acid significantly increased the relaxation of the bioassay ring observed upon stimulation of the endothelial cells with adenosine diphosphate (3 x 10(-8) M to 3 x 10(-4) M) and, to a lesser extent with bradykinin (10(-8) to 3 x 10(-8) M), while the relaxing activity evoked by the calcium ionophore A23187 (3 x 10(-7) M) was not affected. Neither acetylcholine (10(-6) M) nor 5-hydroxytryptamine (10(-6) M) stimulated the release of relaxing factor(s) from control or eicosapentaenoic acid-treated endothelial cells. 4. Bradykinin (10(-7) M), adenosine diphosphate (3 x 10(-5) M), the calcium ionophore A23187 (10(-6) M) and nitric oxide (2 x 10(-6) M) stimulated haemoglobin-sensitive increases in cyclic GMP content of porcine endothelial cells which were unaffected by prior chronic exposure to eicosapentaenoic acid. 5. These results suggest that chronic exposure of porcine aortic endothelial cells to eicosapentaenoic acid increases the release of relaxing factor(s) in response to activation of membrane-associated receptors for purines and kinins. The lack of effect of eicosapentaenoic acid treatment on agonist-stimulated production of cyclic GMP suggests that the enhanced relaxation observed under bioassay conditions is not due to an increased production of nitric oxide.

Increases in Flow Reduce the Release of Endothelium-Derived Relaxing Factor in the Aorta of Normotensive and Spontaneously Hypertensive Rats

Experiments were designed to compare basal and acetylcholine-induced release of endothelium-derived relaxing factor at different flow rates in the aorta of the normotensive (Wistar-Kyoto; WKY) and spontaneously hypertensive (SHR) rat. Aortic segments (with endothelium) of either WKY or SHR were perfused at different flow rates (1 or 4 mL/min) with modified Krebs-Ringer solution; the relaxing activity of the perfusate from the two types of segments was bioassayed by measuring the isometric force in rings (without endothelium, and contracted with norepinephrine) of aortas of both WKY and SHR. All studies were performed in the presence of indomethacin to prevent endothelial production of prostacyclin and other vasodilator prostanoids. The basal release of endothelium-derived relaxing factor was not significantly affected by the flow rate in either the WKY or the SHR aortas. At the two flow rates, and with both types of bioassay rings, the basal release of endothelium-derived relaxing factor was smaller with SHR than with WKY aortas, but this reached significance only at 4 mL/min using the SHR-aorta as bioassay tissue. Both with WKY and SHR aortas the release of endothelium-derived relaxing factor evoked by acetylcholine was significantly larger at 1 than at 4 mL/min; no significant differences in responsiveness to acetylcholine were noted between WKY and SHR segments. There was also no difference in responsiveness of WKY and SHR bioassay rings to acetylcholine and acetylcholine-induced release of endothelium-derived relaxing factor. These experiments suggest that prolonged exposure to increases in shear stress reduces the ability of the endothelium to release endothelium-derived relaxing factor in responses to muscarinic activation.

Canine arteries release two different endothelium-derived relaxing factors.

Experiments were designed to analyze the effects of ouabain on the response of vascular smooth muscle to endothelium-derived relaxing factors released under basal conditions and on stimulation with acetylcholine or bradykinin. Bioassay rings of canine coronary artery (without endothelium) were superfused with perfusate from canine left circumflex coronary arteries with endothelium (donor arteries). During contractions of the bioassay ring evoked by prostaglandin F2 alpha, the relaxations caused by endothelium-derived relaxing factor(s), released under basal conditions or on exposure of the endothelial cells of the donor artery to maximally effective concentrations of acetylcholine, were reduced by incubation of the bioassay ring with ouabain. However, the relaxations evoked by infusion of bradykinin were not altered by incubation of the bioassay rings with ouabain. These experiments demonstrate the release of two endothelium-derived relaxing factors that can be distinguished using ouabain.

Release of different relaxing factors by cultured porcine endothelial cells.

Experiments were performed to determine the effect of ouabain on the release of relaxing factor(s) from cultured endothelial cells, and its action on the effect of the relaxing factor(s) on arterial smooth muscle. A column of porcine aortic endothelial cells grown on microcarrier beads in suspension culture was perfused with modified Krebs-Ringer bicarbonate solution. The release of relaxing factor(s) by the endothelial cells was detected under bioassay conditions by measuring the relaxing activity of the perfusate overflowing a ring of canine coronary artery (without endothelium) contracted with prostaglandin F2 alpha. Incubation of the endothelial cells with ouabain did not affect the relaxation of the bioassay ring under basal conditions or upon stimulation of the endothelial cells with ADP but impaired the relaxation induced by bradykinin or the calcium ionophore A23187. Incubation of the bioassay ring with ouabain reduced the relaxation under basal conditions as well as the relaxation induced by ADP but did not affect the relaxation observed upon stimulation with bradykinin and A23187 and the endothelium-independent relaxations induced by nitric oxide. These experiments suggest that cultured porcine aortic endothelial cells release two endothelium-derived relaxing factors; one is released under basal conditions and upon stimulation with adenosine diphosphate and the other (which presumably is nitric oxide) upon stimulation with bradykinin and the calcium ionophore A23187.