Vasodilator efficacy of nitric oxide depends on mechanisms of intracellular calcium mobilization in mouse aortic smooth muscle cells.

BACKGROUND AND PURPOSE: Reduction of intracellular calcium ([Ca(2+)](i)) in smooth muscle cells (SMCs) is an important mechanism by which nitric oxide (NO) dilates blood vessels. We investigated whether modes of Ca(2+) mobilization during SMC contraction influenced NO efficacy. EXPERIMENTAL APPROACH: Isometric contractions by depolarization (high potassium, K(+)) or alpha-adrenoceptor stimulation (phenylephrine), and relaxations by acetylcholine chloride (ACh), diethylamine NONOate (DEANO) and glyceryl trinitrate (GTN) and SMC [Ca(2+)](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 [Ca(2+)](i), which was suppressed by sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA) inhibition, but not by soluble guanylyl cylase (sGC) inhibition. Relaxation of K(+)-stimulated segments did not parallel a decline of [Ca(2+)](i). However, stimulation (BAY K8644) of L-type Ca(2+) 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 Ca(2+) re-uptake into the SR and was prominent when intracellular Ca(2+) was mobilized. The other involved sGC-stimulated cGMP formation, causing relaxation without changing [Ca(2+)](i), presumably by desensitizing the contractile apparatus. This pathway seems related to L-type Ca(2+) influx, and L-type Ca(2+) channel blockers increase the vasodilator efficacy of NO.

Heterogeneity in relaxation mechanisms in the carotid and the femoral artery of the mouse.

The participation of prostanoids, nitric oxide and non-prostanoid non-nitric oxide factors in endothelium-dependent relaxations was investigated in phenylephrine (PE)-constricted carotid and femoral arteries of C57BL6 mice. The carotid artery was more sensitive to acetylcholine as compared to the femoral artery, and cyclooxygenase inhibition did not influence the relaxation in either vessel. In the carotid artery, high doses of acetylcholine caused transient constrictions, which were abolished by indomethacin or piroxicam. In the carotid but not the femoral artery, N(omega)-nitro-L-arginine or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) enhanced PE-induced contractions enormously, suggesting that endogenous nitric oxide production is much higher in the carotid artery. While in the carotid artery all relaxation was abolished by N(omega)-nitro-L-arginine or ODQ, a residual response (34+/-5% and 74+/-4%, respectively) but with a different shape, was maintained in the femoral artery. This N(omega)-nitro-L-arginine-resistant relaxation was abolished by the combination of apamin and charybdotoxin. In both arteries, ODQ abolished relaxation to S-nitroso-N-acetyl-D-penicillamine, while N(omega)-nitro-L-arginine enhanced the sensitivity to this donor of exogenous nitric oxide. In 30 mM KCl, the relaxation to acetylcholine was abolished by N(omega)-nitro-L-arginine or ODQ in either artery. In conclusion, in the carotid artery endothelium-dependent relaxation is mediated predominantly by nitric oxide acting via cyclic GMP-dependent pathways, while in the femoral artery part of the relaxation can be attributed to a non-prostanoid non-nitric oxide factor operating via apamin/charybdotoxin-sensitive potassium channels.

Exposure to oxidized low-density lipoprotein in vivo enhances intimal thickening and selectively impairs endothelium-dependent dilation in the rabbit.

OBJECTIVES: Based on in vitro studies, oxidized low-density lipoprotein (oxLDL) has been implicated in atherogenesis and the associated deficiency in endothelium-dependent relaxation. The aim of this study was to investigate the effects of in vivo exposure to oxLDL on intimal thickening and relaxing behaviour. METHODS: Intimal thickening was evoked by the placement of silicone collars around the carotid arteries of the rabbit for 3 or 14 days. OxLDL (Cu(2+)-oxidized, 7 micron/h) or the vehicle phosphate-buffered saline (PBS) was infused in the collars via subdermally implanted osmotic minipumps. RESULTS: The collared vessels receiving PBS developed discrete intimal thickening after 14 days (intima/media (I/M) ratio 11 +/- 2%). OxLDL infusion resulted in intimal thickening after 3 days and significantly enhanced the intimal thickness by 14 days (I/M ratio 98 +/- 16%). Collaring alone for 3 or 14 days and 3 days exposure to oxLDL did not impair the endothelium-dependent relaxations to acetylcholine or calcium ionophore, nor to the NO donors glyceryl trinitrate (GTN) and S-nitroso-N-acetylpenicillamine (SNAP). However, the sensitivity to acetylcholine was decreased after exposure to oxLDL for 14 days (-logEC50 oxLDL 6.95 +/- 0.11 vs. 7.52 +/- 0.11 collar alone) and the maximal relaxation to the endothelium-dependent agonist was reduced by 50%, this in the presence of a virtually intact endothelium. Complete relaxation was still obtained with the nitric oxide donors. CONCLUSION: Our results show for the first time that local vascular exposure to oxLDL in vivo promotes intimal thickening and inhibits endothelium-dependent dilation, thereby supporting an active role for oxLDL in the morphological and functional changes observed in atherosclerotic blood vessels.

Local application of LDL promotes intimal thickening in the collared carotid artery of the rabbit.

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 micrograms h-1, n = 16 arteries), oxLDL (Cu2+ oxidized, 7 micrograms h-1, n = 16), or phosphate-buffered saline (5 microL h-1, n = 16). Segments proximal to the collars served as controls. Collar placement without lipoprotein application resulted in the appearance of alpha-SMC actin-immunoreactive cells in the intima, thereby increasing the intimal thickness from 5 +/- 1 to 26 +/- 5 microns. 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 approximately 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.

Dual effects of oxidized low-density lipoprotein on immune-stimulated nitric oxide and prostaglandin release in macrophages.

Oxidized low-density lipoprotein (LDL) is currently regarded as a tentative key player in atherosclerosis by virtue of its ability to induce intracellular lipid accumulation and to modulate cell functions in the vessel wall. We previously demonstrated that inducible nitric oxide (NO) synthase activity is attenuated in lipid-laden J774 macrophages obtained by incubation with oxidized LDL 200 micrograms ml-1 for 24 h. In the present study we investigated the effect of oxidized LDL in a lower concentration (20 micrograms ml-1) or for a shorter time (6 h) and the possible mediator role of prostaglandin E2 and prostacyclin. Prostaglandins and the NO synthase metabolites citrulline and nitrite were elevated in the 24 h supernatant after immune stimulation with interferon-gamma 100 U ml-1 with or without lipopolysaccharide 10 micrograms ml-1. Pretreatment with oxidized LDL 20 micrograms ml-1 for 18 h decreased nitrite release by 31 +/- 2%, whereas prostaglandin production was not affected. A 6 h pre-exposure to 200 micrograms ml-1 had an opposite effect: it significantly potentiated interferon-gamma-stimulated prostaglandin E2 (10-fold), prostacyclin (7-fold), nitrite (1.5-fold), and citrulline (2.4-fold) release. Indomethacin 10 microM abolished the prostaglandin production and largely prevented the oxidized LDL-dependent increase in NO synthase activity. Acetylated LDL was without effect. The data show that the immune-induced release of NO is potentiated or suppressed, depending on the conditions of exposure to oxidized LDL. The potentiation due to short, high-dose exposure is partly mediated by prostaglandins since indomethacin inhibited both processes.

Effects of storage temperature and fetal calf serum on the endothelium of porcine aortic valves.

Endothelial integrity and function may be an important determinant for long-term success of allograft heart valves. To determine the optimal storage temperatures for preservation of long-term endothelial function in porcine aortic valves, different storage temperatures and times were investigated. Fresh valves were either (1) stored at 4 degrees C, with or without 10% fetal calf serum supplement, for 1, 2, 4, 7, 14, 21, or 28 days; (2) cryopreserved for 2, 4, or 8 weeks at -80 degrees C or -170 degrees C; (3) cryopreserved in long-term storage (as long as 1 year), with or without fetal calf serum, at -170 degrees C. Viability of endothelial cells was assessed through measurement of the production of prostacyclin in basal and bradykinin-stimulated conditions, during in vitro incubation of the valve cusps at 37 degrees C. Endothelial morphologic variations in valves stored at 4 degrees C were evaluated by scanning electron microscopy. With storage at 4 degrees C, after 4 days the valves already produced significantly less (p < 0.05) prostacyclin than fresh preparations in both basal (0.21 +/- 0.04 versus 3.56 +/- 0.03 ng.ml-1.cm-2) and stimulated conditions (4.17 +/- 0.36 vs 24.23 +/- 1.83). Morphologic changes could not yet be distinguished with scanning electron microscopy at that time. When the storage period was extended, the levels of prostacyclin further diminished; after 14 days, prostacyclin release could no longer be detected. In cryopreserved valves, prostacyclin production was similar for as long as 2 weeks of storage either at -80 degrees C or at -170 degrees C in basal (2.69 +/- 0.63 vs 2.93 +/- 0.51) and stimulated (16.43 +/- 3.19 vs 16.50 +/- 2.57, = 6) conditions. After 8 weeks, no prostacyclin release could be detected in valves stored at -80 degrees C. After 6 months storage at -170 degrees C, the prostacyclin production was significantly (p < 0.05) reduced compared with fresh valves; it then remained constant for as long as 1 year. The valves stored with fetal calf serum produced significantly (p < 0.05) less prostacyclin than did those without fetal calf serum. For longer cryopreserved banking, we recommend storing heart valves at -170 degrees C instead of at -80 degrees C to maintain viability of endothelial cells. Fetal calf serum would harm endothelial viability during long-term cryopreservation.

Dexamethasone influences intimal thickening and vascular reactivity in the rabbit collared carotid artery.

Intimal thickening predisposes to atherosclerosis and is often associated with alterations of the vascular reactivity of the artery. We investigated whether dexamethasone inhibited the intimal thickening and reactivity changes induced by a silicone collar placed around the left rabbit carotid artery for 2 weeks. The sham-operated, right artery served as control. Dexamethasone (1 mg/kg/day), given in the drinking water (n = 10) or by a subcutaneous minipump (n = 10), abolished intimal thickening compared to that of both placebo groups (n = 10). Both dexamethasone and the collar suppressed the isometric force development of isolated segments elicited by KCl in organ chamber experiments. The collar raised the sensitivity to serotonin (5-hydroxytryptamine, 5-HT) and the maximum force development (Emax) after normalization for the KCl responses. Dexamethasone exerted complex effects on 5-HT contractions in sham arteries: the curves often became biphasic, and sensitivity and Emax of the first phase were depressed by dexamethasone. In contrast, dexamethasone raised the hypersensitivity of collared arteries to 5-HT even further. Collar and dexamethasone did not influence endothelium-dependent relaxations elicited by acetylcholine or the calcium ionophore A-23187. It is concluded that dexamethasone interfered with neo-intima formation in the collar model, presumably by inhibition of smooth muscle cell migration and/or proliferation, without restoring contractile behaviour. Therefore, the collar-induced alterations in the reactivity of the smooth muscle cells in the media appear to be unrelated to the process of intimal thickening.

Thrombin triggers the de novo expression of an inducible NO synthase in porcine aortic valve endothelial cells.

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.

Effects of different antibiotics on the endothelium of the porcine aortic valve.

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, p < 0.05) than the controls (4.66 +/- 0.66--30.55 +/- 3.84 ng/ml x cm2, p < 0.001). 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.

Improved endothelial viability of heart valves cryopreserved by a new technique.

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.

Release and vascular activity of endothelium-derived relaxing factor in atherosclerotic rabbit aorta.

A diet containing 0.3% cholesterol was given to male New Zealand rabbits for 16 weeks; this produced atherosclerotic lesions (fatty streaks) on 80% of the intimal surface of the thoracic aorta and on 45% of the intimal surface of the abdominal aorta. The endothelium-dependent relaxations induced by acetylcholine, substance P and ionophore A23187 were inhibited in the atherosclerotic aortas. Besides the endothelium-independent relaxations induced by nitroglycerine, the relaxations induced by atrial natriuretic peptide (ANF) were also significantly reduced in the more atherosclerotic thoracic aorta. In bioassay experiments it was found that acetylcholine and substance P caused a smaller release of endothelium-derived relaxing factor (EDRF) from atherosclerotic thoracic aortas than from control thoracic aortas: the EDRF released by the vasodilators evoked less relaxation in atherosclerotic detector abdominal aortas than in control detector abdominal aortas. Nitric oxide evoked significantly less transient relaxation in the atherosclerotic thoracic and abdominal aortas than in the respective control tissues. The data indicate that as experimental atherosclerosis in the rabbit progresses, both vascular activity and EDRF release become affected; this leads to a complete loss of endothelium-dependent relaxation in the more atherosclerotic blood vessels.

Effect of hypercholesterolemia on vascular reactivity in the rabbit. II. Influence of treatment with dipyridamole on endothelium-dependent and endothelium-independent responses in isolated aortas of control and hypercholesterolemic rabbits.

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)

Effect of hypercholesterolemia on vascular reactivity in the rabbit. I. Endothelium-dependent and endothelium-independent contractions and relaxations in isolated arteries of control and hypercholesterolemic rabbits.

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.