Phospholipid methylation in rabbit aorta

The formation of phosphatidylcholine by successive methylations of phosphatidylethanolamine using S-adenosylmethionine as the methyl donor was studied in homogenates of rabbit aorta. Addition of phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine, but not phosphatidylethanolamine, stimulated methyltransferase activity and this activity was further stimulated when the phospholipids were dispersed in taurocholate prior to addition to the assay system. No incorporation of radiolabel into sphingomyelin or lysolecithin was detected indicating minimal metabolism of newly formed phosphatidylcholine. The majority of methyltransferase activity was detected in the high-speed pellet of the aortic homogenate; however, since activity was also detected in the high-speed supernatant, the low-speed supernatant preparation was used as the source of enzyme. Methyltransferase activity was characterized in cultured arterial smooth muscle cells using methionine as the radiolabeled precursor. The major product formed was phosphatidylcholine. No difference in enzyme activity was seen as a function of the length of time that cells were in culture or anatomic location of the aortic explant used as a source of cells. Treatment of the cells with cycloheximide did not affect methyltransferase activity. The ability of catecholamine agonists and vasoactive peptides to influence methyltransferase activity was investigated both in the cell-free preparation and in cultured cells. These compounds did not appear to alter methyltransferase activity in rabbit aortic smooth muscle cells.     

Involvement of cyclooxygenase 2 in the protective effect of 17beta-estradiol on hypercholesterolemic rabbit aorta

The involvement of cyclooxygenase (COX) in the effects of 17beta-estradiol was investigated on hypercholesterolemic rabbits aorta. Acetylsalycilic acid, nimesulide, or SQ22536 was used as respective antagonist of COX-1, COX-2, or adenylate cyclase using aortic rings precontracted with phenylephrine and exposed to cumulative concentrations of acetylcholine (ACh). The relaxation effect of ACh was impaired by hypercholesterolemia and restored by an 8-week 17beta-estradiol treatment. In the control group treated with estrogen, nimesulide, acetylsalycilic acid, or SQ22536 slightly reduced the response to ACh. In hypercholesterolemic rabbits treated with estrogen, nimesulide significantly reduced the maximal relaxation and shifted to the right the relaxation curve of ACh, whereas acetylsalycilic acid did not modify the maximal response to ACh but displaced slightly the concentration-response curve. SQ22536 reduced the relaxant effect of ACh down to the level obtained in the presence of nimesulide. These results suggest that the protective effect of 17beta-estradiol against hypercholesterolemia involved COX-2/adenylate cyclase pathway.     

Some characteristics of intimal folds in the rabbit aorta

A stereomicroscopic study of fresh rabbit aorta has shown that the intimal folds form a special pattern which reappears when the aorta is relaxed after stretching. The pattern of intimal folds seems to be adapted to and may be the result of continuously repeated movements of the vessel wall. This supports the view hat intimal folds are present in vivo.     

Passive viscoelastic properties of the rabbit aorta in vitro

Tensile tests on longitudinal and circumferential strips of the rabbit aorta have been performed. Stress-strain and relaxation parameters have been estimated with respect to four stress levels and three positions on the aorta. Stress-strain data indicate that in longitudinal direction the aorta becomes more compliant with distance from the aortic arch. The opposite tendency is found for the circumferential direction. Stress-relaxation is found to be strongly dependent on the stress level. The results are discussed with regard to arterial dynamics.     

Atherogenic effect of Arecoline: A computational study

There are over 600 million people worldwide covering Asian and Oceanic countries including India have the habit of chewing areca nut as masticator in different forms. Arecoline (C(8)H(13)NO(2)) has been reported as one of the abundant constituents of areca nut. A good number of scientific publications have made Arecoline responsible for oral cancer. Based on observation from clinical situation in North East India, one of the most betel quid chewing region of the country, we suspected a link between consumption of areca nut and Cerebro Vascular Disease like stroke. Therefore, we considered Low Density Lipoprotein (LDL) receptor as target and Arecoline as ligand and studied ligand -target interaction using computational tools. Also we considered High Density Lipoprotein (HDL) receptor as another target to see if Arecoline has any binding potential with it over and above LDL receptor. Docking result indicated that Arecoline and Cholesterol both, have affinity towards extracellular domain of Human LDL receptor but affinity of Arecoline is much higher (-12.3560.) than that of Cholesterol(-0.1810). Docking of Arecoline and 1, 2-Hexyl-1- cyclopentanone thiosemicarbazone (thiosemicarbazone) with Bovine HDL receptor showed that Arecoline also has the potential (Score, -6.2690Kcal/Mol) to block HDL receptor though its potential is less than that (score, -10.0509 Kcal/Mol) of control (thiosemicarbazone). We, therefore, suggest that by inhibiting endocytosis of LDL cholesterol because of blocking LDL receptor function and also by preventing LDL cholesterol uptake by liver from blood because of interference with HDL receptor, Arecoline may contribute to atherosclerosis. The study therefore, indicates a positive correlation between chewing of betel quid and Cerebro Vascular Disease.     

Succinate dehydrogenase activity in the wall of rabbit aorta

Summary An investigation of succinate dehydrogenase activity in the wall of rabbit aorta was carried out. The level of succinate dehydrogenase per se in the smooth muscle cells was found to be fairly high, while the mitochondrial level of carrier CoQ was low. The latter may explain the low level or lack of activity of succinate dehydrogenase in these cells as noticed by previous authors.A reliable image of the actual level of succinate dehydrogenase was obtained only by adding CoQ₁₀ to the incubation system. PMS should be avoided, as it induced a Nothing dehydrogenase reaction even at low concentrations.     

Endothelium-dependent relaxation and experimental atherosclerosis in the rabbit aorta

This study was undertaken to determine whether the production or release of the endothelium-dependent relaxatory factor is impaired in atherosclerotic New Zealand White rabbits. Atherosclerosis was induced by feeding a diet containing 2% cholesterol for 6 weeks. The production or release of endothelium-dependent relaxatory factor was assayed as follows. A 5-cm length of aorta donor was perfused with Krebs-bicarbonate buffer and the perfusate drained over a deendothelialized ring of recipient aorta set up for recording isometric tension. The recipient was precontracted with norepinephrine (0.2 mumol/L) in the perfusate. When acetylcholine was added to the perfusate, the recipient relaxed in a dose-dependent manner. This assay was used to compare the relaxatory responses produced in recipient rings by adding acetylcholine to donors from atherosclerotic and control rabbits. The relaxation produced by atherosclerotic donors were smaller than those generated by control donors (16.5 +/- 4.9 vs. 32.7 +/- 5.3%; n = 10, p less than 0.05). It is suggested that in atherosclerotic rabbits the ability of aortic endothelium to produce or release endothelium-dependent relaxatory factor is impaired.     

Biosynthesis of fibronectin by rabbit aorta

The in vitro interactions between vascular cells and fibronectin have been shown to influence phenotypic expression of both cultured endothelial and smooth muscle cells. To more effectively assess the potential functional role of fibronectin in vivo in modulating vascular phenotypes, we have established methodology for studying fibronectin biosynthesis in the rabbit aorta using aortic rings that are morphologically and functionally intact and metabolically active. Aortic rings were incubated with 35S-labeled methionine in a supplemented physiological salt solution. The tissue was fractionated, and quantitative immunoprecipitation was performed using a polyclonal antibody directed against human plasma fibronectin. Newly synthesized fibronectin was most abundant in the fraction solubilized using 4% sodium dodecyl sulfate and in the incubation medium. In all fractions studied, fibronectin was present predominantly as a dimer with no detectable aggregates of fibronectin. Pulse-chase experiments showed that a substantial amount of newly synthesized fibronectin was found in the 4% sodium dodecyl sulfate extract after only 1 h, suggesting that fibronectin was rapidly incorporated into the extracellular matrix. The more soluble forms of newly synthesized fibronectin appeared to be the precursors for secreted fibronectin, and no precursor-product relationship between soluble and insoluble fibronectin was found. Dissection of aortic rings following incubation with labeled methionine showed that newly synthesized fibronectin was uniformally distributed in both intima-media and media-adventitia segments. Endothelial cell denudation caused only a 20% decrease of fibronectin biosynthesis concomitant with similar changes in total protein biosynthesis, consistent with the medial smooth muscle cell as the major source of newly synthesized fibronectin. Biosynthesis of fibronectin was increased following a 24-h preincubation of the aortic rings, and concomitant increases in steady state mRNA for fibronectin were found. These in vitro studies documented the utility of aortic rings for the general purpose of studying protein synthesis in vascular cells and provide new information on the characteristics of fibronectin biosynthesis by aortic tissue.     

Inhibition of lysophospholipase by cholesterol in rabbit aorta

Lysophospholipase activity was measured in rabbit aorta using 1-[1-14C]palmitoyl-sn-glycero-3-phosphocholine as a substrate. The enzyme did not require Ca2+ for its activation and the maximal activation was attained in the presence of EGTA. Cholesterol dose-dependently inhibited the lysophospholipase activity in the soluble fraction and IC50 value was approximately 15 microM. Lineweaver-Burk plot revealed that cholesterol competitively inhibited lysophospholipase and Km values in the presence and absence of cholesterol (15.5 microM) were 12.3 and 2.8 microM, respectively. Vmax values were approximately 475 pmol/min.mg. The results suggest that cholesterol can interact with the enzyme per se, resulting in the inhibition of the lysophospholipase activity in rabbit aorta.     

Characterization of endothelial thromboxane receptors in rabbit aorta

An increased synthesis of thromboxane (TX) A₂ is associated with a number of cardiovascular diseases including atherosclerosis, unstable angina and hypertension. We previously identified a subgroup of NZW rabbits in which isolated arteries failed to contract to the TX agonists, U46619 or I-BOP. In vascular smooth muscle membranes, there was a significant decrease in TX receptors, termed TP. These rabbits are referred to as vTP− and those with the TP receptor are called vTP+. Because TP receptors are expressed in some types of endothelial cells, the present study was designed to determine whether functional TP receptors are present in endothelial cells cultured from aortas of vTP+ and vTP− rabbits. Radioligand binding studies were performed with ¹²⁵I-BOP. Aortic endothelial cells from vTP+ rabbits exhibited specific and saturable binding. In contrast, in endothelial preparations from vTP− rabbit aortas, no measurable binding to ¹²⁵I-BOP was detected. Using an anti-TP receptor antibody, we compared the amount of receptor expressed in endothelial cell lysates obtained from vTP+ and vTP− rabbits. Consistent with the results observed radioligand binding assays, the expression of TP receptor protein was decreased in vTP− compared to vTP+ endothelial cells. An in vitro wound healing assay was used on confluent monolayers of endothelial cells. In the untreated vTP+ cells, the area of the scratch was completely closed by 30 h. In the vTP+ cells treated with U46619 (3 μM), the rate of closure of the scratch area was reduced with approximately 12% of the scratch area remaining at 30 h. Pretreatment with the TP receptor antagonist, SQ 29548 (10 μM) prevented the inhibitory effect of U46619. The rate of closure of the scratch in the vTP− was not altered by U46619. In a separate study, U46619 (3 μM) increased the release of 6-keto PGF_1α, the stable metabolite of prostacyclin, in vTP+ but not vTP− endothelial cells. Pretreatment with SQ29548 (10 μM) or the cyclooxygenase inhibitor, indomethacin (10 μM) blocked the increase in vTP+ endothelial cells. In vascular reactivity studies in aortas from vTP+ rabbits, removal of the endothelium enhanced the vasoconstrictor response to U46619 indicating that activation of endothelial TP receptors may modulate vascular tone via the release of the vasodilator, prostacyclin. The results of this study suggest an important role for endothelial TP receptors in modulating vascular function.     

Characterization of endothelial thromboxane receptors in rabbit aorta

An increased synthesis of thromboxane (TX) A(2) is associated with a number of cardiovascular diseases including atherosclerosis, unstable angina and hypertension. We previously identified a subgroup of NZW rabbits in which isolated arteries failed to contract to the TX agonists, U46619 or I-BOP. In vascular smooth muscle membranes, there was a significant decrease in TX receptors, termed TP. These rabbits are referred to as vTP- and those with the TP receptor are called vTP+. Because TP receptors are expressed in some types of endothelial cells, the present study was designed to determine whether functional TP receptors are present in endothelial cells cultured from aortas of vTP+ and vTP- rabbits. Radioligand binding studies were performed with (125)I-BOP. Aortic endothelial cells from vTP+ rabbits exhibited specific and saturable binding. In contrast, in endothelial preparations from vTP- rabbit aortas, no measurable binding to (125)I-BOP was detected. Using an anti-TP receptor antibody, we compared the amount of receptor expressed in endothelial cell lysates obtained from vTP+ and vTP- rabbits. Consistent with the results observed radioligand binding assays, the expression of TP receptor protein was decreased in vTP- compared to vTP+ endothelial cells. An in vitro wound healing assay was used on confluent monolayers of endothelial cells. In the untreated vTP+ cells, the area of the scratch was completely closed by 30 h. In the vTP+ cells treated with U46619 (3 microM), the rate of closure of the scratch area was reduced with approximately 12% of the scratch area remaining at 30 h. Pretreatment with the TP receptor antagonist, SQ 29548 (10 microM) prevented the inhibitory effect of U46619. The rate of closure of the scratch in the vTP- was not altered by U46619. In a separate study, U46619 (3 microM) increased the release of 6-keto PGF(1alpha), the stable metabolite of prostacyclin, in vTP+ but not vTP- endothelial cells. Pretreatment with SQ29548 (10 microM) or the cyclooxygenase inhibitor, indomethacin (10 microM) blocked the increase in vTP+ endothelial cells. In vascular reactivity studies in aortas from vTP+ rabbits, removal of the endothelium enhanced the vasoconstrictor response to U46619 indicating that activation of endothelial TP receptors may modulate vascular tone via the release of the vasodilator, prostacyclin. The results of this study suggest an important role for endothelial TP receptors in modulating vascular function.     

Fenfluramine-induced thermogenesis in adult domestic fowl (Gallus domesticus): Elimination by propranolol,a beta-blocker

1. Intra-muscular injection of DL-fenfluramine, a 5-hydroxytryptamine agonist, increased heat production by a mean of 16% over the following 6hr in adult domestic fowl.2. Propranolol, a beta-receptor blocker, completely eliminated the effect of fenfluramine on thermogenesis.3. Respiratory quotient (RQ) was significantly reduced by fenfluramine injection, whether or not this was preceded by injection of propranolol. Injection of propranolol alone produced a decline of RQ to 0.71 within 1 hr, followed by an immediate steady increase to 0.90 over the next 5 hr.4. Extension and lowering of the wings, which augments surface area for heat loss, was observed within 30 min of fenfluramine injection and persisted for several hours. This thermolytic effect of fenfluramine was not eliminated by prior injection of propranolol. Polypnea occurred only when both propranolol and fenfluramine had been injected.5. Food intake over the whole day of measurement was significantly reduced by fenfluramine injection, whether or not this had been preceded by injection of propranolol. Water intake over the same period was unaffected by any of the treatments.6. Fenfluramine reduced spontaneous activity by almost half. The reduction was slightly greater when fenfluramine injection followed propranolol. Propranolol given alone had no effect on activity.     

Role of nitric oxide on the vasorelaxant effect of atrial natriuretic peptide on rabbit aorta basal tone

The role of nitric oxide (NO) on the vasorelaxant effect of atrial natriuretic peptide (ANP) on the basal tone of rabbit aortic rings conditioned to angiotensin II (Ang II) was studied. ANP aortic relaxation and nitrite release were measured in the presence and absence of endothelium and a NO-synthase inhibitor. Ang II at 10(-8) M triggered a contractile response, conditioning the vessel to a vasorelaxant effect of ANP (10(-8) M). This effect was significantly enhanced by endothelium removal, NG-nitro-L-arginine methyl ester (L-NAME, 10(-4) M), and methylene blue (10(-5) M). ANP decrease of basal tone in Ang-II-sensitized aortic rings was improved when a higher concentration of Ang II was used (l0(-6) M). Basal and Ang-II-stimulated nitrite release were measured in stretched (S) and nonstretched (NS) aortic rings. Nitrite release was significantly increased in S rings (p < 0.001). L-NAME (10(-4) M) partially inhibited nitrite release in both basal and Ang-II-stimulated S aortic rings. In NS aortic rings, the NO inhibitor did not inhibit basal nitrite release but blunted the Ang-II-stimulated nitrite level. A significant negative correlation between nitrite release and the ANP vasorelaxant effect on basal tone was dependent on the Ang-II-sensitizing dose. The present results demonstrate that ANP relaxant effects on aortic basal tone are related to NO levels, which are regulated by S- and Ang-II-concentration-dependent NO generation and quenching.