Rabbit Aortic Smooth Muscle Cells Research Articles

A comparative study of the metabolic activation of 3,4-benzo[ a]pyrene to mutagens by aorta smooth muscle cells of rat and rabbit

The mutagenic activation of benzo[ a]pyrene (BaP) after exposure to aorta smooth muscle cells of different origin was examined. Three test systems with different genetic endpoints — sister-chromatid exchange (SCE), gene mutation at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) locus and uncheduled DNA synthesis (UDS) — were used. Treatment of rat and rabbit aorta smooth muscle cells with BaP (1–6 μg/ml) resulted in a significant increase of SCEs, HGPRT mutations and UDS. So smooth muscle cells are capable of converting BaP to metabolites with a DNA-damaging action. In order to investigate the relation between the formation of mutagenic BaP metabolites and the susceptibility to atherosclerosis we compared the mutagenic potential of BaP using aorta smooth muscle cells of different species (rat, rabbit) and locations (thoracic and abdominal aorta). Rabbits and abdominal aortas are more susceptible to atherosclerosis than rats and thoracic aortas. The SCE, HGPRT and UDS assays revealed that smooth muscle cells of differnt origin possessed the same metabolic potential towards BaP. There was no correlation between the mutagenic potency of BaP metabolites and the susceptibility to atherosclerosis. As smooth muscle cells have a low metabolic capacity towards BaP, probably other factros in addition to the metabolic capacity of smooth muscle cells are responsible for species and tissue differences in susceptibility to atherosclerosis.

Serum-induced proliferation of rabbit aortic smooth muscle cells from the contractile state is inhibited by 8-Br-CAMP but not 8-Br-cGMP

A method is described for the quantification of vascular smooth muscle cell growth from individual explants of contractile rabbit aortic tunica media. The precision of the method probably depends on regular explant geometry (1-mm squares) and pooling sufficient explants. Serum-induced growth was quantified by measurements of ATP concentration, incorporation of [ 3H]thymidine and DNA concentration. The possible effects of endogenous vasodilator agents on growth were investigated by using lipid soluble analogues of their second messengers, namely 8-Br-cAMP and 8-Br-cGMP, which are known to relax rabbit aortic strips. Cell growth was inhibited concentration-dependently by 8-Br-cAMP but not 8-Br-cGMP (0.01-1 mM). The effect of 8-Br-cAMP was reversible, and also occurred when addition was delayed until after growth had commenced. The results imply that endogenous vasodilators such as prostacyclin, adenosine and adrenaline, which increase cAMP concentration, may normally suppress smooth muscle cell growth, whereas nitric oxide and atriopeptins, which increase cGMP concentration, may not.

Marked acceleration of exogenous fatty acid incorporation into cellular triglycerides by fetuin.

Fetuin belongs to a group of fetal glycoproteins whose specific function is not known. In this study we investigated the effect of bovine fetuin on exogenous fatty acid incorporation into lipid classes by fetal rabbit aortic smooth muscle cells (SMC) and human fetal skin fibroblasts. When compared with albumin, the addition of fetuin to the culture medium caused a dramatic increase in labeled fatty acid incorporation (nanomoles/mg of protein) by SMC into triglycerides (albumin (control) 2.8 +/- 0.3 + fetuin 178.3 +/- 13.7). This effect was noted at a wide range of fetuin concentrations (0.2-5%) at oleate:fetuin molar ratios of 3.3-0.13, respectively. Similar effects were noted using human fetal skin fibroblasts with both labeled oleic and arachidonic acids (0.1 mM) as substrates (arachidonic acid incorporation into triglycerides, albumin (control) 76.9 +/- 16.2 + fetuin 684.6 +/- 64.1). Stimulation of fatty acid incorporation into di- and monoglycerides was also noted. Although the amount of unbound fatty acid in the presence of fetuin was greater than with albumin, experiments done under conditions that create identical unbound oleate levels (by varying fatty acid concentration) still showed increased fatty acid incorporation into triglycerides by SMC when exposed to fetuin. This marked effect of fetuin on triglyceride accumulation in cells was confirmed by lipid analysis, strong positive staining with oil red O, and transmission of electron microscopy. Furthermore, the potential physiological role of fetuin in terms of fatty acid and transport was attested by (a) the presence of significant amounts of free fatty acids associated with fetuin; and (b) by the stimulatory effect of fetuin, even when added to culture media containing other fatty acid carriers. These results show that (a) fetuin is far more efficient than albumin in incorporating fatty acids into cells; and (b) this might represent a novel function for fetuin during development.

The nature of large vessel disease in diabetes mellitus

Reprint requests: Thomas Ledet, MD, Research Laboratory for Biochemical Pathology, Aarhus Kommunehospital, DK-8000, Aarhus C, Denmark. The problem of cardiovascular disease and diabetes mellitus can be subdivided into one distinct zone where large-scale research work has provided a number of well documented pieces of information from epidemiologic and clinical studies, and another, large and indeterminate grey region of unasked questions and unposed problems, which is inhabited by borderline problems between diabetes and cardiovascular research. The epidemiologist has told us that heart disease occurs 3-5 times more frequently among diabetic patients. At present, it is not possible to explain this very high frequency as a consequence of the usual risk factors in atherosclerosis, such as increased serum LDL.’ Likewise, various experimental investigations have not been able to provide an explanation through analyses of non-enzymatic glycosylated lipoproteins.* In our laboratory, the study of large vessel disease in diabetes mellitus is guided by a hypothesis that postulates the existence of a diabetic macroangiopathy which develops independent of the usual atherosclerotic risk factors, but is a result of diabetic intermediary metabolism and endocrinopathy.3s4 Since deposition of a strong PAS-positive substance in the wall of the smaller blood vessels is a characteristic feature of diabetic microangiopathy, we decided to investigate the extent of this PAS-positive substance in the larger coronary arteries from a group of NIDDM patients.5 The data obtained from the diabetic patients were compared with the results from a group of age and gender matched nondiabetic subjects. An area of the tunica media was selected, based on the presence or absence of atherosclerotic plaques in the tunica intima. Using a traditional point counting procedure, we estimated the area occupied by the PAS-positive material in the tunica media. It appeared that a significantly larger area was occupied by the PAS-positive substance in the coronary arteries from diabetic individuals than was the case in nondiabetics. Moreover, it appeared that the size of the PAS-positive area was completely independent of the presence of atherosclerotic plaques (Figure 1). The same pattern was observed when the amount of collagen was evaluated using a histologic staining technique and the same point counting. In other histologic sections taken from the same samples of the coronary arteries, the area occupied by acid mucopolysaccharides (glycosaminoglycans) was evaluated by identification with alcian blue, again with the point counting procedure. It could be shown that the area containing acid mucopolysaccharides was substantially reduced in coronary arteries from diabetic patients, independent of the presence of atherosclerotic plaques. The basement membrane in the tunica media is a PAS-positive structure surrounding individual arterial smooth muscle cells.6s7 Since the basement membrane is abnormal in many organs in diabetic patients, we have focused on the elaboration of that structure in a tissue culture system of rabbit aortic smooth muscle cells. Using normolipemic fasting serum from a group with IDDM and a group with NIDDM, we have evaluated the elaboration of basement membrane, and found that sera from the diabetic patients significantly enhanced the accumulation of basement membrane material compared with sera from normolipemic nondiabetic individuals of the same age and gender (Figure 2).* In the same culture system, we tested the effect of hyperlipemic serum obtained from rabbits fed a high cholesterol diet for more than 6 weeks, but found no influence on basement membrane accumulation.

Glycosaminoglycan synthesis by smooth muscle cells of differing phenotype and their response to endothelial cell conditioned medium

The synthesis of glycosaminoglycans (GAG) by contractile and irreversible synthetic phenotypes of vascular smooth muscle cells (SMC), and their response to endothelial cell conditioned medium (ECCM), has been investigated. Contractile SMC, (with a high volume fraction of myofilaments) were obtained by culturing freshly isolated rabbit aortic SMC for 3 days in primary culture. Irreversible synthetic SMC (with a low volume fraction of myofilaments) were obtained by serially passaging SMC to achieve more than 5 cumulative population doublings. In fresh medium both phenotypes produced significant amounts of GAG, but irreversible synthetic cells were more than twice as active on a per cell and cell volume basis. The proportions of individual GAG also changed with change in phenotype. Hyaluronic acid (HA) was the predominant GAG (78%) synthesised by contractile SMC but was significantly reduced (47%) in the irreversible synthetic cells with a corresponding increase in sulphated GAG (SGAG). The changed levels in GAG synthesis were independent of SMC growth. Both phenotypes responded to ECCM from bovine endothelial cells (EC) and significantly increased their synthesis of GAG and by the same relative amounts (50–100%). This response was density dependent, with ECCM from low and high density cultures of EC producing maximal responses and EC of intermediate densities producing minimal increases. Furthermore, dense cultures of EC preferentially stimulated SGAG. These findings show that an increase in synthesis of SMC GAG, and especially sulphated GAG as is found in atherosclerosis, may occur either through a change in phenotype or through endothelial mediated stimulation of GAG synthesis by either phenotype.

The stimulatory effect of heavy metal cations on proliferation of aortic smooth muscle cells.

Heavy metal cations Cd2+, Pb2+, and Hg2+ were added to substitute for Ca2+ in culture media to study their effect on the relationship between CaM and the proliferation of cultured rabbit aortic smooth muscle cells (ASMC). It was found that all the heavy metal cations studied stimulated the proliferation of ASMC in varying degrees, increased the CaM content in cells at late G1 stage and decreased the activity of cAMP PDE. These results suggest that the adverse effect of heavy metals may be related to the pathogenesis of atherosclerosis and hypertensive disease.

Collagen synthesis by cultured rabbit aortic smooth-muscle cells. Alteration with phenotype

Enzymically isolated rabbit aortic smooth-muscle cells (SMC) in the first few days of primary culture express a 'contractile phenotype', but with time these cells modulate to a 'synthetic phenotype'. Synthetic-state SMC are able to proliferate, and, provided that they undergo fewer than 5 cumulative population doublings, return to the contractile phenotype after reaching confluency [Campbell, Kocher, Skalli, Gabbiani & Campbell (1989) Arteriosclerosis 9, 633-643]. The present study has determined the synthesis of collagen, at the protein and mRNA levels, by cultured SMC as they undergo a change in phenotypic state. The results show that, upon modulating to the synthetic phenotype, SMC synthesized 25-30 times more collagen than did contractile cells. At the same time, non-collagen-protein synthesis increased only 5-6-fold, indicating a specific stimulation of collagen synthesis. Steady-state mRNA levels are also elevated, with alpha 2(I) and alpha 1(III) mRNA levels 30 times and 20 times higher respectively, probably reflecting increased transcriptional activity. Phenotypic modulation was also associated with an alteration in the relative proportions of type I and III collagens synthesized, contractile SMC synthesizing 78.1 +/- 3.6% (mean +/- S.D.) type I collagen and 17.5 +/- 4.7% type III collagen, and synthetic cells synthesizing 90.3 +/- 2.0% type I collagen and 5.8% +/- 1.8% type III collagen. Enrichment of type I collagen was similarly noted at the mRNA level. On return to the contractile state, at confluency, collagen production and the percentage of type I collagen decreased. This further illustrates the close association between the phenotypic state of SMC and their collagen-biosynthetic phenotype.

Cell-mediated contraction of collagen lattices in serum-free medium: Effect of serum and nonserum factors

This study was conducted to identify a defined, serum-free culture medium that supports cell dependent contraction of a collagen lattice. Collagen lattices were found to contract in cultures containing human foreskin fibroblasts (HFF) or rabbit aortic smooth muscle (RASM) cells incubated in serum-free medium. HFF and RASM cells required different supplements to contract the collagen gels. HFF cultured in Dulbecco's modified Eagle's (DME) medium supplemented with bovine serum albumin (BSA) and either endothelial cell growth supplement (EnGS), insulin (In), or platelet derived growth factor (PDGF) supported collagen lattice contraction. Replacement of BSA with casein without the addition of other supplements improved contraction. In contrast, RASM cells supplemented with BSA, EnGS, In, and PDGF were able to contract collagen gels only minimally. Similar to HFF, RASM cells cultured in DME medium supplemented with casein, but without the addition of other supplements, contracted collagen lattices. HFF-mediated collagen contraction was inhibited by prostaglandins E1 or E2, fibronectin, or ascorbic acid. The reported serum-free model provides a useful in vitro method to investigate the role of serum and nonserum factors regulating cell mediated-contraction of insoluble collagen fibrils.

Antiproliferative action of cyclic GMP-elevating vasodilators in cultured rabbit aortic smooth muscle cells

In cultured rabbit aortic smooth muscle cells (SMCs), sodium nitroprusside (SNP) (10 −7 to 10 −4 M), atrial natriuretic peptide (ANP) (10 −9 to 10 −6 M) and 8-bromo-cyclic GMP (10 −6 to 10 −3 M) inhibited the whole blood serum (WBS)-induced DNA synthesis by about 30%. The doses of SNP and ANP necessary for the inhibition of the WBS-induced DNA synthesis were similar to those necessary for the formation of cellular cyclic GMP (cGMP). These agents were effective even when added 6 h after stimulation of the cells with WBS. These results suggest that cGMP inhibits the proliferation of rabbit aortic SMCs by inhibiting the progression from the G 1 into S phase of the cell cycle and raise the possibility that cGMP-elevating vasodilators may suppress the atherogenic process by inhibiting vascular SMC proliferation.

Phorbol myristate inhanced specific incorporation of arachidonic acid into phospholipids through lysophospholipid acyltransferase in cultured smooth muscle cells

The effect of stimulation of phospholipase with phorbol 12-myristate 13-acetate and lipopolysaccharide on 1-acyl-glycerophospholipid acyltransferase was studied in cultured rabbit aorta smooth muscle cells. The acyltransferase in smooth muscle cells without stimulation was active on a wide range of unsaturated fatty acids and was not arachidonic acid specific. Upon increase in phospholipase activity, acyltransferase activity only with arachidonic acid as substrate increased in a time-dependent fashion. Apparent acyltransferase activity was increased most upon increase in phospholipase activity when lysophosphatidylcholine was used as acceptor. These results suggest that arachidonic acid specific acyltransferase was induced in smooth muscle cells by increase in phospholipase activity. The role of this acyltransferase is postulated to be the specific incorporation of endogenously released arachidonic acid.

A variant derived from rabbit aortic smooth muscle: phenotype modulation and restoration of smooth muscle characteristics in cells in culture.

We examined the relationship between growth arrest of smooth muscle cells and structural changes in microfilament bundles, and also that between the structural changes and the actions of contractile agonist using a multipassagable variant cell line (SM-3) derived from rabbit aortic smooth muscle cells. The content of smooth muscle type alpha-actin increased with density-dependent growth arrest of the SM-3 cells, but was attenuated in the logarithmically growing cultures. As assessed cytochemically, the growth-arrested cells contained longitudinally oriented bundles of actin-containing microfilament and myosin-based filaments visualized with rhodamine-phalloidin and antibody against myosin light chain 20, respectively, whereas both actin- and myosin-containing structures in logarithmically growing cells showed slight, shortened, or diffused patterns. Electron microscopic examination of the growth-arrested cells revealed that the cells contained numerous and conspicuous microfilament bundles associated with many compact electron-dense bodies. In addition, pinocytotic vesicles were often found near the plasma membrane in the growth-arrested cells. SM-3 cells in the growth-arrested phase responded to prostaglandin F2 alpha (3-30 microM) and rat endothelin (0.1-1.0 microM) with a reversible contractile response, in association with monophosphorylation and/or diphosphorylation of the myosin light chain 20. However, the influence of the contractile agonists was greatly reduced during logarithmic growth. These results suggest that in the SM-3 cells in the growth-arrested phase, there is a restoration of the contractile architecture and the myosin light chain phosphorylation system. Thus, this SM-3 cell line is expected to serve as a useful model for examining biochemical and physiological phenomena of smooth muscle.

Characterization of a continuous smooth muscle cell line derived from rabbit aorta.

A spontaneously arising continuous cell line (Rb-1) derived from collagenase-elastase digested rabbit aorta has been propagated in vitro for over 100 passages. During this period, the Rb-1 cells remained spindle-shaped and formed regularly oriented parallel bundles. After Passage 50, Rb-1 cells were found to be serum-independent in their growth and reached higher saturation density than rabbit aorta smooth muscle cells. Alpha-actin and desmin filaments were detected by immunostaining in Rb-1 cells and early passage of rabbit aorta smooth muscle cells. The proportion of alpha-actin transcripts in Rb-1 cells was lower than that of transcripts for beta- and gamma-actins. The modal chromosome number was maintained at 44 between Passages 11 and 60, and two marker chromosomes were constantly present. Infection of Rb-1 cells with two strains of herpes simplex virus type 1 resulted in high titers of virus, whereas a herpes simplex virus type 2 temperature-sensitive mutant replicated only at the permissive temperature. The Rb-1 cell line could be used for the study of vascular smooth muscle cell proliferation and their interaction with viruses.

Endothelin-1 increases intracellular calcium mobilization but not calcium uptake in rabbit vascular smooth muscle cells

Conflicting evidence has been reported regarding the role of endothelin-1, a potent vasoconstrictor peptide, in stimulating extracellular calcium influx in rabbit vascular smooth muscle. The objective of this study was to elucidate the effects of endothelin-1 on transmembrane 45Ca 2+ influx and intracellular calcium mobilization in cultured rabbit aortic smooth muscle cells. In calcium containing buffer, endothelin-1 induced a concentration-dependent 45Ca 2+ efflux response over the range of 10 pM to 100 nM with an EC 50 of approximately 60 pM. Maximum endothelin-stimulated 45Ca 2+ efflux was not affected by the absence of extracellular calcium or the presence of 1 μM verapamil. Endothelin-1 did not induce transplasmalemmal 45Ca 2+ uptake at times up to 30 min. These findings suggest that an alteration in intracellular calcium handling, rather than extracellular calcium influx, is responsible for the endothelin-stimulated increase in intracellular calcium concentration in rabbit aortic smooth muscle cells.

Eicosanoid metabolism in cholesterol-enriched arterial smooth muscle cells: reduced arachidonate release with concomitant decrease in cyclooxygenase products.

A biochemical correlation between vascular cholesterol metabolism and eicosanoid biosynthesis has not been fully elucidated. To assess the effects of cholesteryl ester (CE) accretion on eicosanoid synthesis, we studied eicosanoid metabolism in cultured rabbit aortic smooth muscle cells (SMC) following lipid-enrichment by incubation with cationized LDL (cLDL). SMC exposed to cLDL synthesized 50% less immunoreactive 6-keto-PGF1 alpha than untreated cells when exposed to the calcium ionophore, A-23187. In addition, cLDL-treatment reduced arachidonate acid (AA)-induced prostacyclin (PGI2) production sevenfold. Components of cLDL decreased eicosanoid biosynthesis in the following rank-order: linoleate greater than cholesterol greater than apo-cLDL. Lipid-enriched cells incorporated amounts of [1-14C]AA into phosphatidylcholine and phosphatidylethanolamine equal to control cells, but subsequent exposure to ionophore released significantly less radioactivity as free arachidonate (AA), with proportionally less conversion to eicosanoids. Ionophore released equivalent amounts of AA from all phospholipids, suggesting specificity for uptake, but not release of AA by cellular phospholipases. Cells enriched in CE had an eightfold decrease in percentage of phospholipid-derived AA relative to linoleate as compared to controls. Taken together, our data demonstrate that SMC metabolism of cLDL leads to cholesterol and CE accretion concomitant with diminished production of eicosanoids. Potential mechanisms for this effect include competitive inhibition of eicosanoid production by linoleate derived from LDL, direct inhibition of phospholipase A2 activity by cholesterol, and decrease in cyclooxygenase activity. These findings may have pathophysiological significance in that a reduction in PGI2 synthetic capacity of arterial SMC may exacerbate CE deposition since PGI2 promotes intracellular CE hydrolysis.

Relative specificities of inhibition of acid cholesteryl ester hydrolase and neutral cholesteryl ester hydrolase in cultured rabbit aortic smooth muscle cells by esterastin and cholesteryl oleyl ether

Rabbit aortic smooth muscle cells in culture were incubated with 0.04–500 M esterasim. Acid cholesteryl ester hydrolase (ACEH) and neutral cholesteryl ester hydrolase (NCEH) activities were inhibited to a comparable degree, with 50% inhibition occurring in the range of 0.4 M esterastin. Cells incubated with cholesteryl oleyl ether showed 50% inhibition of NCEH at 5.0 M, but no inhibition of ACEH over a concentration range of 0.2–20 M. This relative specificity of cholesteryl oleyl ether for NCEH can be employed to study the relative roles of ACEH vs NCEH in preventing cellular cholesteryl ester accumulation.

Effect of normolipemic and hyperlipemic serum on biosynthetic response to cyclic stretching of aortic smooth muscle cells.

Arterial smooth muscle cells synthesize matrix macromolecules in response to mechanical stimulation. Exposure to serum lipids also stimulates connective tissue fiber accumulation. To assess the effect of serum lipids on the biosynthetic response to tensile stress, we subjected rabbit aortic smooth muscle cells that were cultured on purified elastin membranes to cyclic stretching and relaxation 50 times per minute in the presence of serum-free medium (SFM), normolipemic serum (NLS), or hyperlipemic serum (HLS). Incorporation of 14C-proline into proline and into hydroxyproline was taken as a measure of protein and collagen synthesis. When cells were grown in plastic Petri dishes, exposure to NLS or HLS increased both protein and collagen production to the same extent compared to synthesis in SFM (1.7 times for NLS and 1.6 times for HLS; p less than 0.001 compared to SFM). For cells grown on stationary elastin membranes, NLS and HLS also increased protein and collagen synthesis compared to SFM. The effect of NLS was 1.35 times that of HLS for protein and 1.43 times greater for collagen (p less than 0.03). Cyclic stretching in SFM doubled synthesis for both protein (p less than 0.002) and collagen (p less than 0.002) compared to stationary controls, but had no effect on synthesis in NLS. In HLS, however, cyclic stretching elevated synthesis to the same level as was found in NLS (p less than 0.003). We conclude that the relative inhibition of synthesis on stationary membranes by HLS was not due to a toxic effect, since HLS increased synthesis both in Petri dishes and on elastin membranes, and the amplifying effect of cyclic stretching in HLS was similar to that seen in SFM.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of smooth muscle cell derived growth factor (SDGF) in combination with other growth factors on smooth muscle cells

Recently intimal thickening was shown to be due to stimulation of proliferation of arterial smooth muscle cells (SMC) by autocrine secretion of growth factor(s). We have reported that cultured rabbit aortic SMC secrete a growth factor (SDGF) distinct from other known growth factors. This paper reports on studies on the biological characteristics of SDGF. Putative “competence” and “progression” growth factors synergistically stimulated DNA synthesis of cultured rabbit aortic SMC. Conditioned medium (CM) from SMC containing SDGF stimulated DNA synthesis in SMC synergistically with either the competence factors or the progression factors. This synergistic effect was also observed in the presence of optimal concentrations of both competence and progression factors. The continuous presence of CM was essential for its stimulation of DNA synthesis whereas the presence of PDGF for only the first 4 h of culture was sufficient to induce maximum stimulation of DNA synthesis. These results suggest that SDGF is a new growth factor distinct from either competence or progression factors and that it stimulates a different pathway in SMC from those stimulated by other known growth factors.

Two types of protein kinase C with different functions in cultured rabbit aortic smooth muscle cells

Incubation of cultured rabbit aortic smooth muscle cells (SMC) with phorbol-12,13-dibutyrate (PDBu) for 48 h caused the down-regulation of protein kinase C (PKC) to the level of 30–40% of that in the control cells. The proliferative and antiproliferative actions of PKC were abolished in parallel with the loss of the down-regulation-sensitive component of PKC, but the inhibitory actions in the whole blood serum (WBS)-induced phospholipase C (PLC) reactions and intracellular Ca 2+ mobilization were not affected. Immunoblot analysis with specific monoclonal antibodies against three PKC isozymes (type I, II and III) revealed that only the type III isozyme was detected in rabbit aortic SMC and that this isozyme completely disappeared after the incubation with PDBu. These results indicate that the type III isozyme is responsible for the proliferative and antiproliferative actions and suggest that the unidentified isozyme(s) is involved in the inhibitory actions in the WBS-induced PLC reactions and intracellular Ca 2+ mobilization in rabbit aortic SMC.

Plasmenylethanolamine is the major storage depot for arachidonic acid in rabbit vascular smooth muscle and is rapidly hydrolyzed after angiotensin II stimulation.

The present study demonstrates that rabbit aortic intimal smooth muscle cells contain the majority of their endogenous arachidonic acid mass in plasmenylethanolamine molecular species. To demonstrate the potential significance of these plasmenylethanolamines as substrates for the smooth muscle cell phospholipases that are activated during agonist stimulation, aortic rings were prelabeled with [3H]arachidonic acid and stimulated with angiotensin II. Although the specific activities of the choline and inositol glycerophospholipid pools were similar after the labeling interval, ethanolamine glycerophospholipids had a specific activity of only 20% of the specific activity of choline and inositol glycerophospholipids. Despite the marked disparity in the specific activities of these three phospholipid classes, angiotensin II stimulation resulted in similar fractional losses (35-41%) of [3H]arachidonic acid from vascular smooth muscle choline, ethanolamine, and inositol glycerophospholipid classes. Reverse-phase HPLC demonstrated that greater than 60% of the [3H]arachidonic acid released from ethanolamine glycerophospholipids after angiotensin II stimulation originated from plasmenylethanolamine molecular species. Taken together, the results demonstrate that the major phospholipid storage depot for arachidonic acid in vascular smooth muscle cells are plasmenylethanolamine molecular species which are important substrates for the phospholipase(s) that are activated during agonist stimulation.

Prostacyclin-mediated effect of high density lipoproteins as cellular cholesterol acceptors on aortic smooth muscle cells

Low (LDL) and high (HDL) density lipoproteins stimulate prostacyclin (PGI 2) synthesis in cultured rabbit and human aortic smooth muscle cells. In this respect, the efficacy of HDL exceeded that of LDL, HDL 3 being the most effective. HDL 3 obtained from hypoalphacholesterolemic patients' serum had a lesser stimulative effect on PGI 2 synthesis as compared with HDL 3 of normolipidemic subjects. Partially purified apoprotein A-1 stimulates the metabolism of 14C-arachidonic acid accompanied with enhanced formation of prostaglandins, especially 6-keto-PGI 1α. Within a 24 h incubation in the fetal calf serum-free medium, prostaglandins I 2 and E 1 (1 × 10 −7 m) reduce the intracellular cholesterol level in human aortic smooth muscle cells by 30%. Total HDL fraction as well as HDL 3 and HDL 2b applied in combination with prostaglandins have a synergistic effect resulting in a 50% fall in intracellular cholesterol. Hypothetically, the uptake of cholesterol by HDL may include the following stages: (1) HDL interacts with the cell and activates the intracellular PGI 2 synthesis probably via apo A-1 modulatory action on arachidonic acid metabolism; (2) newly synthesized PGI 2 activates cholesteryl ester hydrolase leading to the formation of free cholesterol; (3) HDL takes up free cholesterol.