Impairment of endothelium-dependent relaxation and changes in levels of cyclic GMP in aorta from streptozotocin-induced diabetic rats.

1. Acetylcholine (ACh)-induced relaxation of aortic strips with endothelium and production of cyclic GMP between streptozotocin-induced diabetic and age-matched control rats were compared. 2. The concentration-response curve for ACh-induced relaxation was shifted to the right in diabetic rats. IC50 values for ACh were 4.57 +/- 0.67 x 10(-8) M and 1.00 +/- 0.87 x 10(-7) M in aortic strips from age-matched control and diabetic rats, respectively (n = 6, P less than 0.05). 3. Relaxations produced by atrial natriuretic peptide (ANP) in diabetic aortae were similar to those in age-matched vessels. 4. Relaxations produced by sodium nitroprusside (SNP) in diabetic aortae were similar to those in age-matched vessels. 5. Basal levels of cyclic GMP and ACh-induced production of cyclic GMP were significantly decreased in diabetic rats. 6. These results suggest that functional changes in endothelium but not in guanylate cyclase activity in the aorta may occur in diabetes, and thus, spontaneous and ACh-induced formation of cyclic GMP may be decreased. This decrease in production of cyclic GMP may be responsible for the decreased response of the aorta to the relaxant effect of ACh.     

Attenuation of endothelium-dependent relaxation in aorta from diabetic rats

Endothelium-dependent relaxation was examined in aortic ring preparations obtained from rats with streptozotocin-induced diabetes. The endothelium-dependent relaxation which was produced by acetylcholine and histamine in aortic rings precontracted with norepinephrine was significantly attenuated in aortic rings from diabetic rats when compared with the relaxation in rings from age-matched control animals. However, the relaxation induced by sodium nitroprusside (an endothelium-independent relaxant agent) in diabetic preparations was comparable to the control. These results show that diabetes leads to an impairment of the endothelium-dependent relaxation of aorta.     

Impairment of endothelium-dependent relaxation in aortae from spontaneously diabetic rats

1. Diabetes mellitus is known to produce alterations in vascular reactivity. The present study examined the effects of endothelium-dependent and endothelium-independent relaxing substances on thoracic aorta from control and spontaneously diabetic rats. 2. Endothelium-dependent relaxation produced by acetylcholine or the calcium ionophore, A23187, in aortic rings precontracted with phenylephrine was significantly attenuated in diabetic vessels. 3. Relaxations produced by sodium nitroprusside or adenosine in diabetic preparations were comparable to those in control vessels. 4. The results show that diabetes leads to a specific impairment of endothelial-dependent relaxation.     

Drug actions in rats with arteriosclerosis induced by toxic doses of vitamin D2.

The vascular response of isolated perfused hind legs from normal and arteriosclerotic rats to noradrenaline, ATP, PGF2 alpha and vasopressin was determined. The increase of perfusion pressure to noradrenaline and ATP was reduced and that to PGF2 alpha and vasopressin was enhanced in arteriosclerotic rats in comparison to normal animals. The results indicate that the reactivity of the vascular smooth muscle of isolated hind legs of arterioslerotic rats is not only quantitatively but also qualitatively different in comparison with normal rats.     

Contractions induced by phenylephrine and noradrenaline are differently affected by endothelium-dependent relaxation in rat aorta.

In rings of rat aorta precontracted with phenylephrine (10 microM) or noradrenaline (10 microM), addition of carbachol (10 microM) produced an endothelium-dependent relaxation. However, regardless of the concentration of agonist tested, both the intensity and duration of the relaxation were significantly less when noradrenaline, rather than phenylephrine, was used as the precontracting agent. The different responses observed do not appear to be related to destruction of endothelium-derived relaxing factor by autoxidation of noradrenaline since neither EDTA (30 microM) nor superoxide dismutase (30 units mL-1) improved the relaxation to carbachol. In addition, in endothelium-free rings, the noradrenaline (1 microM)-induced contraction was less sensitive than the phenylephrine (1 microM)-induced contraction to sodium nitroprusside (0.1 microM) or to 8-Br-cGMP (300 microM). With phenylephrine-, but not noradrenaline-, induced contraction, the relaxation triggered by carbachol was significantly reduced by pretreatment of the aortic rings with chloroethylclonidine (50 microM), which inactivates a subpopulation of alpha 1-adrenoceptors. Thus, the results confirm that both alkylation sensitive and resistant alpha 1-adrenoceptors exist in rat aorta and indicate that EDRF may discriminate between these two alpha 1-adrenoceptor subtypes which are differently affected by phenylephrine and noradrenaline.     

A possible mechanism of endothelium-dependent relaxation induced by pirarubicin and carbachol in rat isolated aorta.

The mechanism of endothelium-dependent relaxation induced by pirarubicin, (2'R)-4'-O-tetrahydropyranyladriamycin, THP, or carbachol was investigated in the rat isolated aorta. The relaxant effect of THP (1.5 x 10(-6)-4.5 x 10(-5) M) or carbachol (10(-8)-10(-4) M) on the aorta with endothelium was decreased by lowering Ca2+ in the medium. The relaxation induced by THP was not inhibited by pretreatment with verapamil (10(-6)-10(-5) M), and that induced by carbachol was only partially inhibited. However, on replacement of all but 20 mM Na+ with either Li+ or choline, the THP- or carbachol-induced relaxation was inhibited. Furthermore, the relaxing effect of THP or carbachol was inhibited by pretreatment with amiloride (10(-4)-3 x 10(-4) M), with ouabain (10(-4)-10(-3) M), or with K(+)-depletion. These results suggest that the THP- or carbachol-induced relaxation depending on endothelium was affected by modifying the calcium ion concentration, and that a Na(+)-Ca2+ exchange process is involved.     

Effect of pravastatin on impaired endothelium-dependent relaxation induced by lysophosphatidylcholine in rat aorta

Aim: To investigate the effects of pravastatin, a potent 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on impaired endothelium-dependent relaxation induced by lysophosphatidylcholine (LPC), the major component of oxidized low-density lipoprotein, in rat thoracic aorta. Methods: Both the endothelium-dependent relaxation response to acetylcholine and the endotheliumindependent relaxation response to sodium nitroprusside of aortic rings were measured by recording isometric tension after the rings were exposed to LPC in the absence or presence of pravastatin to estimate the injury effect of LPC and the protective effect of pravastatin on the aortic endothelium, respectively. Results: Exposure of aortic rings to LPC (1-10μmol/L) for 30 min induced a significant concentration-dependent inhibition of endothelium-dependent relaxation to acetylcholine, but did not affect endothelium-independent relaxation in response to sodium nitroprusside. Pre-incubation of aortic rings with pravastatin (0.3-3mmol/L) for 15 min and then co-incubation of the rings with LPC (3 μmol/L) for another 30 min significantly attenuated the inhibition of endothelium-dependent relaxation induced by LPC. This protective effect of pravastatin (1 mmol/L) was abolished by N^G-nitro-L-arginine methyl ester (30 μmol/L), an inhibitor of nitric oxide synthase, but not by indomethacin (10 μmol/L), an inhibitor of cyclooxygenase. Moreover, protein kinase C inhibitor chelerythrine (1μmol/L) the superoxide anion scavenger superoxide dismutase (200 kU/L), and the nitric oxide precursor L-arginine (3 mmol/L) also improved the impaired endotheliumdependent relaxation induced by LPC, similar to the effects of pravastatin.C onclusion: Pravastatin can protect the endothelium against functional injury induced by LPC in rat aorta, a fact which is related to increasing nitric oxide bioavailability.     

Impairment of pulmonary endothelium-dependent relaxation in patients with Eisenmenger''s syndrome.

A comparison has been made between the endothelium-dependent relaxation of pulmonary arteries (PA) obtained at heart-lung transplantation from 4 patients with Eisenmenger's syndrome and secondary pulmonary hypertension, and PA obtained at lobectomy from 4 patients with lung carcinoma, the controls. All vascular rings were studied immediately after lung excision. PA rings from control patients dose-dependently relaxed to cumulative doses of acetylcholine (ACh, 10(-10) to 10(-5) M), achieving a maximal relaxation of 80 +/- 5% (mean +/- s.e. mean) from precontraction with phenylephrine. By contrast, PA rings from Eisenmenger's syndrome patients achieved a maximal relaxation of only 34 +/- 12% (P less than 0.05, unpaired t test), with even paradoxical contraction at high doses of ACh (10(-6) to 10(-5) M). Sodium nitroprusside (10(-4) M) relaxed all PA rings, with and without endothelium (carefully removed before study), obtained from both control and Eisenmenger's syndrome patients. These results provide the first evidence that endothelium-dependent relaxation of PA mediated by endothelium-derived relaxing factors is impaired in Eisenmenger's syndrome patients with secondary pulmonary hypertension.     

Captopril restores endothelium-dependent relaxation induced by advanced oxidation protein products in rat aorta

To explore whether advanced oxidation protein products (AOPP) can cause endothelial dysfunction in vitro, and whether captopril exerts beneficial effect on impaired endothelium-dependent relaxation induced by exogenous advanced oxidation protein products and to investigate the potential mechanisms. Both the Acetylcholine (ACh)-induced endothelium-dependent relaxation (EDR), sodium nitroprusside-induced endothelium-independent relaxation of aortic rings were measured by recording isometric tension after the rings were exposed to AOPP-BSA in the absence or presence of captopril to assess the injury effect of AOPP-BSA and the protective effect of captopril on the aortic endothelium, respectively. Co-incubation of aortic rings with AOPP-BSA (3 mmol/L) for 90 minutes resulted in a significant inhibition of EDR to ACh, but had no effects on endothelium-independent relaxation to SNP. After incubation of the rings in the co-presence of captopril (3 to 30 micromol/L) or enalaprilat (30 micromol/L) with AOPP-BSA (3 mmol/L) for 90 minutes, captopril significantly and enalaprilat only partly attenuated the inhibition of EDR induced by AOPP-BSA. This protective effect of captopril (30 micromol/L) was abolished by N-nitro-L-arginine methyl ester (10 micromol/L), an inhibitor of nitric oxide synthase. Furthermore, the superoxide anion scavenger superoxide dismutase (SOD, 200 U/mL), and the nitric oxide precursor L-arginine (3 mmol/L) also ameliorated the impaired EDR caused by AOPP-BSA. But D-arginine had no effect on the impaired EDR caused by AOPP-BSA. AOPP-BSA can trigger endothelial dysfunction and captopril can protect the endothelium against functional damage induced by AOPP-BSA in rat aorta, increase nitric oxide bioavailability. The mechanisms of endothelial dysfunction induced by AOPP-BSA may include the decrease of NO and the generation of oxygen-free radicals.     

Involvement of nitric oxide in the endothelium-dependent relaxation induced by hydrogen peroxide in the rabbit aorta.

1. The effects of hydrogen peroxide (H2O2, 0.1-1 mM) on the tone of the rings of rabbit aorta precontracted with phenylephrine (0.2-0.3 microM) were studied. 2. H2O2 induced a concentration-dependent relaxation of both the intact and endothelium-denuded rings. However, in the presence of intact endothelium, H2O2-induced responses were 2-3 fold larger than in its absence, demonstrating the existence of endothelium-independent and endothelium-dependent components of the vasorelaxant action of H2O2. 3. The endothelium-dependent component of H2O2-induced relaxation was prevented by NG-nitro-L-arginine methyl ester (L-NAME, 30 microM) or NG-monomethyl-L-arginine (300 microM), inhibitors of nitric oxide synthase (NOS), in a manner that was reversible by L-, but not by D-arginine (2mM). The inhibitors of NOS did not affect the responses of denuded rings. 4. Methylene blue (10 microM), an inhibitor of soluble guanylate cyclase, blocked H2O2-induced relaxation of both the intact and denuded rings. 5. H2O2 (1 mM) enhanced the efflux of cyclic GMP from both the endothelium-intact and denuded rings. The effect of H2O2 was 4 fold greater in the presence of intact endothelium and this endothelium-dependent component was abolished after the inhibition of NOS by L-NAME (30 microM). 6. In contrast to the effects of H2O2, the vasorelaxant action of stable organic peroxides, tert-butyl hydroperoxide or cumene hydroperoxide, did not have an endothelium-dependent component. Moreover, they did not potentiate the efflux of cyclic GMP from the rings of rabbit aorta.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cholesterol metabolism in serum and aorta of inbred mice fed a high-cholesterol diet

Biochemical characterization of the serum and aorta in inbred C57BL/6Cr mice fed a high-cholesterol diet was investigated by determining the total cholesterol (TC) and free cholesterol (FC) levels in serum, high density lipoprotein (HDL) and aorta. Serum lecithin:cholesterol acyltransferase (LCAT) activity was also determined. A modified fluoroenzymatic method for microdetermination of cholesterol was successfully used. TC and FC levels of the aorta in the mice were significantly increased by the high-cholesterol diet. Serum TC and FC levels of mice fed the high-cholesterol diet were increased about 80% and 110%, respectively, compared with the control. On the other hand, both HDL-TC and HDL-FC levels were decreased about 50%. The HDL-TC/serum-TC ratio was markedly decreased, while the atherogenic index was markedly increased with the high-cholesterol diet. LCAT activity was also strikingly decreased. A positive correlation was observed between LCAT activity and HDL-cholesterol. These changes in the serum may facilitate cholesterol accumulation in the aorta. The results indicate that a biochemical approach using mice may be possible for drug evaluation.     

氨基胍对糖基化蛋白损伤大鼠血管内皮功能的影响

目的　探讨糖基化终末产物形成抑制药氨基胍对外源性制备的糖基化终末产物损伤大鼠离体胸主动脉环内皮依赖性舒张功能的影响及其机制。方法　采用外源性制备的糖基化牛血清白蛋白孵育大鼠离体胸主动脉环 60min诱导血管内皮损伤 ,观察氨基胍对糖基化牛血清白蛋白所致的血管内皮依赖性舒张反应损伤是否具有保护作用。结果　外源性糖基化牛血清白蛋白明显抑制乙酰胆碱诱导的内皮依赖性血管舒张反应 ,但并不影响硝普钠诱导的内皮非依赖性血管舒张反应。用氨基胍 (50～ 50 0 μmol·L- 1 )预孵育血管环 1 5min ,再与糖基化牛血清白蛋白共同孵育 60min ,呈浓度依赖性降低糖基化终末产物对血管内皮依赖性舒张反应的抑制。此外 ,氧自由基清除剂超氧化物歧化酶 (superox idedismutase,SOD ;2× 1 0 5U·L- 1 )也能完全取消糖基化终末产物的抑制作用 ,并与 50 0 μmol·L- 1 氨基胍的保护作用相似。氨基胍 (50 0 μmol·L- 1 )亦能完全逆转SOD抑制剂二乙基二硫氨甲酸酯 (diethyldithiocarbamate,DETC ;1 0 μmol·L- 1 )诱导产生内源性氧自由基所致的血管内皮依赖性反应的损害。结论　氨基胍能取消糖基化终末产物所致大鼠离体胸主动脉环内皮依赖性舒张反应的抑制 ,氨基胍的这种保护作用可能与其抗氧化作用有关     

Abolition of endothelium-dependent relaxation in the rat aorta by tetraoctylammonium ions

Quaternary ammonium ions are common pharmacological probes used to study the kinetic properties of K+ channels in smooth muscle cells. On the other hand, some ammonium compounds cause vasorelaxation through unknown mechanisms. The main aim of this study was to examine a unique role of endothelium in the vascular response to tetraoctylammonium ions (TOA+) in the isolated rat aorta. Changes in contractile force were measured by force transducers and total tissue content of cGMP was measured by radioimmunoassay. Endothelial cytosolic Ca2+ ([Ca2+]i) was assessed by laser scanning confocal microscopy.     

Proteomic analysis of rat aorta during atherosclerosis induced by high cholesterol diet and injection of vitamin D3

1. Atherosclerosis (AS) in rats displays important clinical similarities to human AS. 2. After the experimental model of AS in rat was established and using a proteomic approach, we compared the protein profiling of aorta tissues from healthy and AS rats. 3. Using two-dimensional electrophoresis (2-DE), over 1878 protein species were separated; among them, 1239 protein spots were matched between different gels with average matching rate of approximately 66%. Gel analysis and protein characterization have identified 58 protein spots whose abundance is significantly altered in AS rats. 4. By using matrix-associated laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS) and NCBInr database, 46 proteins were successfully identified. Among them, 18 proteins were of increased abundance in diseased tissues including a group of oxidization-related enzymes such as peroxiredoxin2 and NADH dehydrogenase Fe-S protein 6, components of inflammatory pathways such as lamin A, while 28 proteins were of decreased abundance in the diseased state, including CaM-KII inhibitory protein, transferring, fructose-bisphosphate aldolase. 5. We believe that these results would give insights into the cellular and molecular mechanisms involved in AS development and might lead to the discovery of novel diagnostic markers and new therapeutic opportunities.     

Effects of hesperidin on the progression of hypercholesterolemia and fatty liver induced by high-cholesterol diet in rats

The protective effects of hesperidin against hypercholesterolemia and fatty liver were examined in male Wistar rats fed a high-cholesterol diet for 12 weeks. Compared with a standard diet, a high-cholesterol diet not only increased body weights, liver weights, and serum concentration of cholesterol, but also induced the fatty degeneration (steatosis) of liver. Hesperidin (0.08%) reduced levels of hepatic steatosis, adipose tissue and liver weights (P < 0.05), serum total cholesterol and retinol binding protein (RBP) 4 concentrations (P < 0.05) in rats fed with high-cholesterol diet, while reduction in low-density lipoprotein cholesterol levels and triglyceride concentrations was not significant. It also attenuated the marked changes in mRNA expression of lipid metabolism-related proteins: RBP, heart fatty acid-binding protein (H-FABP), and cutaneous fatty acid-binding protein (C-FABP), in liver and adipose tissue. According to the results of gas chromatography, serum concentrations of total cholesterol and biomarkers of cholesterol synthesis (lathosterol) and absorption (campesterol, β-sitosterol) were lower, and concentrations of cholesterol in feces were higher in the rats given hesperidin (P < 0.05). Hesperidin may improve hypercholesterolemia and fatty liver by inhibiting both the synthesis and absorption of cholesterol and regulating the expression of mRNA for RBP, C-FABP, and H-FABP.     

Impairment of endothelium-dependent relaxation and changes in levels of cyclic GMP in carotid arteries from stroke-prone spontaneously hypertensive rats

Endothelium-dependent relaxation of carotid arteries and changes in levels of cyclic (c)GMP between stroke-prone spontaneously hypertensive (SHRSP) and Wistar-Kyoto (WKY) rats have been compared. The concentration-response curve for acetylcholine (ACh)-induced relaxation was shifted to the right in carotid arteries from SHRSP. Relaxation responses produced by calcimycin (A 23187) and melittin, both endothelium-dependent agents, were depressed in carotid arteries from SHRSP. Relaxation responses produced by sodium nitroprusside and 8-Br-cGMP were similar to those in strips from WKY. ACh-induced production of cGMP was significantly decreased in carotid arteries from SHRSP when compared with the level for similarly treated strips from WKY. These results suggest that functional changes in endothelium, but not guanylate cyclase activity or cGMP sensitivity in the carotid arteries, may occur in hypertension. Thus, impaired endothelium-dependent relaxation in SHRSP may play an important role in hypertensive vascular diseases such as stroke.     

Cryptostrobin and catechin isolated from Eugenia mattosii D. Legrand leaves induce endothelium-dependent and independent relaxation in spontaneously hypertensive rat aorta

BackgroundConsidering the therapeutic potential of phenolic compounds, the purpose of the present study was to investigate the mechanisms involved in the relaxation induced by cryptostrobin and catechin, isolated from Eugenia mattosii D. Legrand leaves, in the aorta of spontaneously hypertensive rats (SHR).MethodsThe thoracic aorta was isolated from SHR and kept in the organ bath system by recording contractile or relaxant responses.ResultsThe addition of cumulative concentrations of cryptostrobin and catechin induced endothelium-dependent and-independent relaxation in aorta rings from SHR, as well as both compounds were effective in reducing phenylephrine-induced contraction. Pretreatment of aortic rings with N_ω-nitro-l-arginine methylester (L-NAME, an inhibitor of nitric oxide synthase) or 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, an inhibitor of soluble guanylate cyclase), resulted in a significant change of relaxant effect induced by catechin, and a slight influence on cryptostrobin-induced relaxation. Muscarinic receptor and potassium channels are involved in catechin-induced relaxation as assessed using atropine (a muscarinic receptor antagonist), tetraethylammonium (a non-selective K⁺ channel blocker) and glibenclamide (an ATP-sensitive K⁺ channel blocker). Conversely, cryptostrobin, but not catechin, blunted the contraction induced by the addition of phenylephrine in a calcium-free solution. Besides that, cryptostrobin attenuated the contraction of rat aorta rings induced by internal Ca²⁺ release and external Ca²⁺ influx.ConclusionsThese findings indicated that cryptostrobin and catechin alter vascular smooth muscle reactivity, and this effect may be involved, at least in part, by enhancing the endothelium NO/cGMP pathway and potassium channels activation. In addition, cryptostrobin reduced the phenylephrine, KCl and CaCl₂-induced contractions in a calcium-free solution.     

Impairment of endothelium-dependent relaxation in the arteries cultured with fetal bovine serum

Effects of chronic treatment with fetal bovine serum on the function of vascular endothelium were examined using an organ culture system. In the rabbit mesenteric arteries cultured with 10% fetal bovine serum for 7 days, the substance P- or ionomycin-induced endothelium-dependent relaxation was significantly attenuated compared to the arteries cultured in the serum-free condition. The effects of the serum were concentration- and time-dependent. By the treatment with the serum, the amounts of nitric oxide (NO) production and total mRNA for endothelial NO synthase were reduced, whereas the sodium nitroprusside-induced relaxation was rather augmented. These results suggest that chronic treatment of rabbit mesenteric artery with fetal bovine serum decreases endothelial NO synthase mRNA, reduces NO production and impairs endothelium-dependent relaxation.     

Impairment of endothelium-dependent relaxation of rat aortas by homocysteine thiolactone and attenuation by captopril

To explore the effects of angiotensin-converting enzyme (ACE) inhibitors on endothelial dysfunction induced by homocysteine thiolactone (HTL). Both endothelium-dependent relaxation and nondependent relaxation of thoracic aortic rings in rats induced by acetylcholine (Ach) or sodium nitroprusside (SNP) and biochemical parameters including malondialdehyde (MDA) and nitric oxide (NO) were measured in rat isolated aorta. Exposure of aortic rings to HTL (3 to 30 mM) for 90 minutes made a significant inhibition of endothelium-dependent relaxation induced by Ach, decreased contents of NO, and increased MDA concentration in aortic tissue. After incubation of aortic rings with captopril (0.003 to 0.03 mM) attenuated the inhibition of endothelium-dependent relaxation (EDR) and significantly resisted the decrease of NO content and elevation of MDA concentration caused by HTL (30 mmol/L) in aortic tissues, a similarly protective effect was observed when the aortic rings were incubated with both N-acetylcysteine (0.05 mM). Treatment with enalaprilat (0.003 to 0.01 mM) made no significant difference with the HTL (30 mM) group regarding EDR, but enalaprilat (0.03 mM) and losartan (0.03 mM) could partly restore the EDR in response to HTL (30 mM). Captopril was more effective than enalaprilat and losartan in attenuation of the inhibition of on acetylcholine-stimulated aortic relaxation by HTL in the same concentration. Moreover, superoxide dismutase (SOD, 200 U/mL), which is a scavenger of superoxide anions, apocynin (0.03 mM), which is an inhibitor of NADPH oxidase, and l-Arginine (3 mmol/L), a precursor of nitric oxide (NO), could reduce HTL (30 mM)-induced inhibition of EDR. After pretreatment with not only the NO synthase inhibitor Nomega-nitro-l-arginine methyl ester (L-NAME, 0.01 mM) but also the free sulfhydryl group blocking agent p-hydroxymercurybenzoate (PHMB, 0.05 mM) could abolish the protection of captopril and N-acetylcysteine, respectively. These results suggest that mechanisms of endothelial dysfunction induced by HTL may include the decrease of NO and the generation of oxygen free radicals and that captopril can restore the inhibition of EDR induced by HTL in isolated rat aorta, which may be related to scavenging oxygen free radicals and may be sulfhydryl-dependent.     

Absence of effect of calcium antagonists on endothelium-dependent relaxation in rabbit aorta.

The effect of chronic feeding of New Zealand White rabbits with nicardipine (60 mg kg-1 daily for 5 weeks) on the endothelium-dependent relaxation (EDR) to acetylcholine (ACh) was examined in vitro. The effect of acute exposure to nicardipine and diltiazem (10 mumol l-1) in the tissue bath was also examined. A bioassay system for endothelium-dependent relaxation factor (EDRF) in which a rabbit aortic ring with endothelium removed was used as recipient and a segment of rabbit aorta with endothelium as donor (producing EDRF in response to ACh) was developed. This system enabled the effect of nicardipine on the synthesis/release and on the relaxation to EDRF to be studied separately. The maximum relaxations to ACh in control and nicardipine-fed animals were 43.6 +/- 5.5 and 53.8 +/- 6.7% (mean +/- s.e. mean) of the contractile response to noradrenaline (NA, 1 mumol l-1) (n = 6, P greater than 0.05). Similarly the EDR to ACh was not significantly altered by acute exposure (30 min) to nicardipine or diltiazem. The maximum relaxations without and with nicardipine were 32.4 +/- 4.2% and 28.0 +/- 3.1% of the contraction to NA (1 mumol l-1) (n = 11, P greater than 0.05). The corresponding data for diltiazem were 42.1 +/- 5.7 and 36.4 +/- 7.3% respectively (n = 11, P greater than 0.05). Both calcium antagonists inhibited the contraction induced by potassium (100 mmol l-1). Nicardipine and diltiazem in concentrations of 100 mumol l-1 reduced the potassium-induced contraction to 33.0 +/- 9.0% and 53.8 +/- 6.7% of control respectively (n = 6, P less than 0.05). In the bioassay experiments the infusion of nicardipine on (a) the recipient tissue only and (b) the donor and the recipient tissue had no significant effect on the relaxant response observed in the recipient tissue when superfused with Krebs-bicarbonate buffer containing ACh via the donor tissue (n = 6, P greater than 0.05). These results indicate that nicardipine and diltiazem had no significant effect on synthesis/release and the relaxant response to EDRF in the rabbit aorta. Thus the translocation of Ca2+ accompanying the EDR to ACh in the rabbit aorta is likely to utilize Ca2+ channels not blocked by these calcium antagonists.