Search

Background and purpose: Reduction of intracellular calcium ([Ca2+]i) in smooth muscle cells (SMCs) is an important mechanism by which nitric oxide (NO) dilates blood vessels. We investigated whether modes of Ca2+ mobilization during SMC contraction influenced NO efficacy. Experimental approach: Isometric contractions by depolarization (high potassium, K+) or α-adrenoceptor stimulation (phenylephrine), and relaxations by acetylcholine chloride (ACh), diethylamine NONOate (DEANO) and glyceryl trinitrate (GTN) and SMC [Ca2+]i (Fura-2) were measured in aortic segments from C57Bl6 mice. Key results: Phenylephrine-constricted segments were more sensitive to endothelium-derived (ACh) or exogenous (DEANO, GTN) NO than segments contracted by high K+ solutions. The greater sensitivity of phenylephrine-stimulated segments was independent of the amount of pre-contraction, the source of NO or the resting potential of SMCs. It coincided with a significant decrease of [Ca2+]i, which was suppressed by sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA) inhibition, but not by soluble guanylyl cylase (sGC) inhibition. Relaxation of K+-stimulated segments did not parallel a decline of [Ca2+]i. However, stimulation (BAY K8644) of L-type Ca2+ influx diminished, while inhibition (nifedipine, 1–100 nM) augmented the relaxing capacity of NO. Conclusions and implications: In mouse aorta, NO induced relaxation via two pathways. One mechanism involved a non-cGMP-dependent stimulation of SERCA, causing Ca2+ re-uptake into the SR and was prominent when intracellular Ca2+ was mobilized. The other involved sGC-stimulated cGMP formation, causing relaxation without changing [Ca2+]i, presumably by desensitizing the contractile apparatus. This pathway seems related to L-type Ca2+ influx, and L-type Ca2+ channel blockers increase the vasodilator efficacy of NO.

The nitric oxide (NO) production by porcine aortic valve endothelial cells was estimated in cusps incubated at 37 degrees C by measuring their cyclic GMP content and the nitrite levels of the incubation medium. After a stabilization period, incubation for 5 min with acetylcholine, bradykinin, ADP and bovine thrombin resulted in a receptor-mediated increase in cyclic GMP which could be blocked by EGTA, N-omega-nitro-L-arginine methyl ester (L-NAME) and NG-monomethyl-L-arginine (L-NMMA). Incubation with lipopolysaccharide (endotoxin) from E. coli O111:B4 or bovine for 5 h, dose-dependently increased nitrite production as well as cyclic GMP content. The elevated nitrite production was completely abolished in the presence of the protein synthesis inhibitor cycloheximide, was reduced by more than 50% by dexamethasone but was not affected by EGTA. L-NMMA dose-dependently reduced the increased nitrite production and cyclic GMP content. These results suggest that besides the presence of a constitutive NO synthase in porcine aortic valve endothelial cells thrombin, like lipopolysaccharide, triggers the de novo expression of an inducible Ca(2+)-independent NO synthase.

Abstract Oxidized LDL (oxLDL) has been implicated in atherogenesis on the basis of in vitro studies and is present in atherosclerotic lesions. The aim of this study was to investigate the effects of LDL and oxLDL on intimal thickening in vivo. Intimal thickening was evoked by the placement of silicone collars around the carotid arteries of rabbits for 2 weeks. The collars were connected to osmotic minipumps containing LDL (7 μg h −1 , n=16 arteries), oxLDL (Cu 2+ oxidized, 7 μg h −1 , n=16), or phosphate-buffered saline (5 μL h −1 , n=16). Segments proximal to the collars served as controls. Collar placement without lipoprotein application resulted in the appearance of α-SMC actin–immunoreactive cells in the intima, thereby increasing the intimal thickness from 5±1 to 26±5 μm. The perivascular infusion of LDL or oxLDL within the collar significantly enhanced the development of the intima ninefold and sevenfold, respectively. The large intimas resulting from lipoprotein exposure were infiltrated by macrophages and T lymphocytes, and the intimal collagen area was increased from 5±2% in the discrete collar-induced intima to ≈20% in the lipoprotein-evoked lesions. In conclusion, the local vascular application of LDL, oxidized in vitro or possibly in vivo, elicited an inflammatory-fibroproliferative response characteristic of arteriosclerotic lesions, thereby demonstrating an active role for this class of lipoproteins in the disease process.

Homograft heart valves are usually sterilized by exposure to multiple-antibiotic solutions at 4 degrees C for 24 hours. Several combinations of antibiotics have been proposed and discussed in the literature, but their toxicity to cusp endothelium has not been investigated yet. We studied the endothelial cell viability of porcine aortic valves by measuring their in vitro prostacyclin (PGI2) production after being exposed to different antibiotics solutions. Porcine aortic valves were immersed for 24 hours at 4 degrees C in RPMI medium to which antibiotics (Gentamycin 80 micrograms/ml, Azlocillin 500 micrograms/ml, Cloxacillin 25 micrograms/ml, Metronidazole 100 micrograms/ml, Amphotericin B 50 micrograms/ml, GACMA) were added separately or in combination. The basal and bradykinin (10 microM) stimulated PGI2 release of these valves in the medium were measured during consecutive incubation lasting 15 minutes at 37 degrees C, using a radioimmunoassay for 6-oxo-PGF1 alpha. Valves treated with the combination of all five antibiotics produced significantly less PGI2 in basal and stimulated conditions (1.64 +/- 0.63--7.25 +/- 1.73 ng/ml x cm2, p0.05) than the controls (4.66 +/- 0.66--30.55 +/- 3.84 ng/ml x cm2, p0.001). Although all antibiotics, when studied separately at the above mentioned concentrations, tended to reduce the biosynthesis of PGI2, amphotericin B was responsible for the most pronounced decrease in its production. The toxic effect of amphotericin B was dose dependent; at a low concentration (5 micrograms/ml), which is usually enough for antifungal action, toxicity was undetectable. At 50 micrograms/ml PGI2 production was half of that found at 5 micrograms/ml, although concentrations as high as 100 micrograms/ml have been used clinically to disinfect homografts. Scanning electron microscopy (SEM) also confirmed the extensive loss of endothelium after exposure to high concentrations of amphotericin B. Our study suggests that the other four antibiotics used in the concentrations described above do not damage endothelial function; amphotericin B is also harmless if used at a concentration of 5 micrograms/ml.

The aim of this study was to compare different techniques of aortic valve cryopreservation by studying the viability of the endothelial cells. Viability was assessed by measuring their in vitro prostacyclin (PGI2) production under basal and stimulated conditions. Fresh and cryopreserved porcine valves were incubated at 37 degrees C in tissue culture medium and PGI2 content in the medium was measured every 15 min up to 300 min. Cryopreservation by the older procedure A included 5% fetal calf serum (FCS) in the preservation medium, a plastic box inside a freezing plastic bag, a cooling schedule approximating -2 degrees C/min, a long thawing time and few dilution steps of the cryoprotectant dimethylsulphoxide (DMSO). The newer procedure B differed from A in packaging, freezing and thawing rates and DMSO dilution. Procedure C was similar to B with the exception that FCS was omitted. Leaflets preserved by procedure A produced significantly less prostacyclin as compared to those treated according to procedures B or C. We conclude that minor differences in the cryopreservation method can become critical to endothelial functional viability.

The present study investigated whether lesion-free aorta segments of young apoE-/- mice, which will later show endothelial dysfunction when atherosclerotic lesions develop, display normal endothelial function in comparison with ‘wild-type’ (wt) C57Bl/6J mice. Endothelial function was assessed by studying endothelium-dependent relaxation to acetylcholine (ACh) and concomitant elevations of endothelial internal calcium (Ca2+i). Therefore, atherosclerosis-prone aorta strips were mounted in a myograph with the endothelial side down and loaded with fura2-AM (340/380 excitation emission ratio in relative units, RU) to measure Ca2+i simultaneously with isometric force. In 90% of WT females (n = 10), 31% of WT males (n = 13), 100% of apoE-/- females (n = 14) and 82% of apoE-/- males (n = 11), relaxation of precontracted (1 μM phenylephrine, PE) strips by ACh (2 × 10−6 M) was accompanied by a contemporary increase of Ca2+i. ACh-induced Ca2+i increase was not different between WT and apoE-/- mice, but was greater in females than in males (0.14 ± 0.03 RU vs. 0.04 ± 0.03 RU, P

The present study investigated whether the higher contractile behaviour of apoE-/- compared with wild type (wt) C57Bl/6J aortic segments is due to an increased contractile status of the smooth muscle cells or a lower basal relaxant effect of the endothelium. Endothelium-intact or -denuded aortic strips or rings were mounted in a myograph and loaded with fura2-AM to measure internal calcium (Ca2+i) simultaneously with isometric force. In endothelium-intact segments, basal Ca2+i was significantly lower in apoE-/- females (n = 7) than in wt females (n = 8), wt males (n = 8) or apoE-/- males (n = 7), i.e. 0.40 ± 0.04 vs. 0.73 ± 0.06, 0.77 ± 0.07 and 0.66 ± 0.09 relative units (340/380 excitation emission ratio). Remarkably, isometric force evoked by 1 μM phenylephrine (PE) was significantly higher in apoE-/- females than in wt females, wt males or apoE-/- males, i.e. 0.59 ± 0.06 vs. 0.18 ± 0.04, 0.20 ± 0.05, and 0.20 ± 0.06 mN/mm, whereas the concomitant PE-induced Ca2+i increase was similar in all groups. Incubation of apoE-/- strips with NOsynthase inhibitors (300 μM L-NNA + L-NAME) increased force in females from 0.61 ± 0.19 to 0.80 ± 0.22 (n = 5) mN/mm (+131%) and in males from 0.37 ± 0.09 to 0.78 ± 0.12 (n = 4) mN/mm (+210%). Experiments in endothelium-denuded aortic rings revealed that the contractile status of smooth muscle cells in wt and apoE-/- females was not altered, whereas contractility of apoE-/- male rings was even reduced in comparison with wt males. It is concluded that in comparison with wt mice, reduced basal NO levels and not a higher contractile status of the smooth muscle cells is responsible for the higher contractile behaviour of apoE-/- females. In males, the contractile status of the smooth muscle cells seemed to be compromised, but not the basal endothelial cell function.

A selected ion monitoring method for the determination of 6-oxo-PGF1 alpha, the stable end-product of prostacyclin, in biological fluids has been developed. In this method, biosynthetically prepared [2H6]-6-oxo-PGF1 alpha is used as internal standard. The method involves extraction, thin-layer chromatography purification and derivatization into the methyl ester, methoxime, trimethylsilyl ether derivatives by carrying out the methoximation first. Quantitative gas chromatographic mass spectrometric analysis is performed in the electron impact mode by monitoring the [M - (TMSOH + CH3O)]+ fragment ions. The use of this method in the measurement of 6-oxo-PGF1 alpha in serous fluids and in incubation media of serous tissues is described.

The effects of cholesterol-feeding in the presence of dipyridamole (0.60 g daily) on contractile responses and on endothelium-dependent and endothelium-independent relaxations in isolated rabbit aortas are described. The investigations were performed simultaneously with those described in Part I (Circ Res 1986; 58:552-564), where the effects of cholesterol feeding on vascular reactivity in rabbit arteries (n = 8 in each group) selected at random from the same group of animals was studied. In the hypercholesterolemic rabbits treated with dipyridamole for 8 or 16 weeks, both the increases in plasma cholesterol and the formation of fatty streaks were significantly less pronounced than in the hypercholesterolemic rabbits not receiving the drug. Segments of the isolated arteries were mounted in organ chambers for isometric tension recording. The contractions caused by acetylcholine, prostaglandin F2 alpha, norepinephrine, clonidine, and serotonin and the endothelium-independent relaxations to nitroglycerin were not significantly altered by the hypercholesterolemia in rabbits treated with dipyridamole, even after 16 weeks of treatment. Thus, the decreased responses to norepinephrine, clonidine, and nitroglycerin and the augmented responses to serotonin noted in aortas of hypercholesterolemic rabbits in Part I were absent in the dipyridamole-treated hypercholesterolemic animals. The endothelium-dependent relaxations to ATP and acetylcholine were not affected after 8 weeks of hypercholesterolemia in presence of dipyridamole, while after 16 weeks the relaxations to ATP and acetylcholine were attenuated only in the more severely affected arteries. The effects of hypercholesterolemia + dipyridamole on endothelium-dependent relaxations were significantly less pronounced than those induced by hypercholesterolemia alone.(ABSTRACT TRUNCATED AT 250 WORDS)

We studied the effects of hypercholesterolemia on vascular responsiveness in different arteries isolated from rabbits: control groups of rabbits and groups receiving the atherogenic diet consisted of eight animals each. In the arteries, 16 weeks of cholesterol-rich (0.3%) diet evoked intimal lesions which were more pronounced than those noted after 8 weeks of hypercholesterolemia; the aortic arch was affected significantly more by the lesions than the abdominal aorta and the pulmonary artery. Segments of the arteries were mounted in organ chambers for isometric tension recording or for measurement of the endothelium-derived relaxant factor. Contractions caused by acetylcholine and prostaglandin F2 alpha were not altered by the hypercholesterolemia; those evoked by serotonin were moderately augmented only in the aortic arch of hypercholesterolemic rabbits. As the degree of intimal lesion formation increased, the contractions to norepinephrine and clonidine were progressively inhibited. The endothelium-independent relaxations to nitroglycerin were inhibited in only the most severely affected arteries; the endothelium-dependent relaxations to acetylcholine and adenosine triphosphate were progressively inhibited as the degree of fatty streak formation augmented. Thus, in the aortic arch, the relaxations to 3 X 10(-6) M acetylcholine, expressed as percent of the initial contraction, decreased from 86.7 +/- 3.3% in control tissues to 16.3 +/- 8.6% in the 16-week hypercholesterolemic vessels; in the abdominal aortas these relaxations averaged 93.5 +/- 2.2% in control vessels and 72.0 +/- 6.9% in the hypercholesterolemic tissues. The acetylcholine-induced release of endothelium-derived relaxant factor from the abdominal aorta was not significantly affected by the hypercholesterolemia. We conclude from these studies that in arteries obtained from hypercholesterolemic rabbits: the contractions caused by serotonergic mechanisms tend to be augmented, while those to alpha-adrenergic activation are decreased, the endothelium-independent relaxations are modified only in the more severely affected arteries, and the endothelium-dependent relaxations are progressively inhibited as the degree of fatty streak formation augments, probably because a step subsequent to the release of endothelium-derived relaxant factor is altered.

Endothelium may release EDRF and PGI2; the former causes relaxation of arterial smooth muscle cells, whereas PGI2 suppresses phagocyte and platelet activation and may relax some arteries. The aim of the present experiments was to investigate whether EDRF from rabbit aorta affects platelet reactivity. To this end the isolated thoracic aorta was perfused with a Krebs' solution (3 ml/min), andthe perfusate was continuously super fused over a de-endothe-lialized ring of the abdominal aorta, contracted withnoradrenaline in the presence of atropine, to monitor EDRF. Aliquots (200 μl) ofeffluent were also added to 200 (il prewarmedautologous citrate platelet rich plasma, which was immediately stimulated with ADP or U46619, a thromboxane (TX) A2 mimetic. Both agonists induce aggregation independently of TXA2 formation. Finally, the PGI2 content of the effluent was assessed by specific radioimmunoassay of 6-oxo-PGF1alpha(ir-6oxo). Infusion of 0.3,1 and 3 μM acetylcholine (Ach) through the aorta for 7.5 min caused a dose-dependent relaxation of the abdominal ring (33 to 100 % at 3 (μM Ach), indicating intraluminal EDRF release. Maximum relaxation was reached in 3 min and maintained. Release of platelet inhibitory activity was variable. At 3 μM Ach, anti-platelet activity(more than 10 % suppression of aggregation)was present in the effluent of 5 (of 7) aorta's for ADP-, and in effluent of 6 (of 7) aorta's for U46617-induced aggregation. U46619 was more sensitive to the anti-platelet activity, a finding consistent withauthentic PGI2. When a clear anti-platelet activity was monitored, its release was abolished by indo-methacin, which affected neither EDRF release nor platelet aggregation. Baseline release of ir-6oxo was low and variable, but dose-dependently stimulated by Ach, reaching a maximum for each concentration in the 2nd to 3rd minute. In contrast to EDRF, ir-6oxo release was not maintained during the subsequent 4 min and tapered off. Formation of ir-6oxo and anti-aggregatingeffect were positively correlated, but therewas no association between EDRF and ir-6oxo or anti-platelet activity. In conclusion, rabbit aortic endothelium could release EDRF and PGI2, but the present set-up did not reveal an anti-platelet effect of EDRF.