Evolution of coronary vascular tone in vasospastic angina.

In general, anginal symptoms diminish with time in patients with vasospastic angina. We assessed changes in coronary vascular tone (CVT) in patients with vasospastic angina over a 4-year period to evaluate the time course of spastic activity. Vasospastic angina was evaluated in 39 patients in whom occlusive spasm was evoked by selective intracoronary injection of ergonovine maleate (ERG-S) 48 h after stopping all coronary vasodilator drugs. These patients had no organic coronary stenosis and developed no stenosis during follow-up. ERG-S was repeated 3 times at 2-year intervals. CVT was determined at each ERG-S study using the equation: CVT = 1-(coronary artery diameter before ERG-S/coronary artery diameter after intracoronary injection of isosorbide dinitrate). Thirty-four patients (87%) had no angina pectoris at the second ERG-S study and 28 (72%) had none at the third. Coronary spasm was induced in 25 patients at the second ERG-S study and in 20 at the third. The overall CVT was shown to decrease at each successive ERG-S study (p < 0.01). There was no correlation between changes in CVT and anginal symptoms or the coronary spasm induction rate. These results demonstrate that CVT decreases over time in patients with vasospastic angina, suggesting that myocardial ischemia may improve spontaneously.

Pharmacological heterogeneity of constrictions mediated by endothelin receptors in rat pulmonary arteries.

The endothelin (ET) receptors mediating rat pulmonary arterial constrictions were investigated. ET-1 and ET-3 constricted both isolated intrapulmonary artery (IPA) and extrapulmonary artery (EPA), with ET-1 having a potency approximately 10 times that of ET-3. The ET(B) selective agonist IRL-1620 produced constriction of only IPA. The ET(B) selective antagonist BQ-788 suppressed the ET-1-induced constriction of IPA only. The ET(A) selective antagonist BQ-123 more effectively antagonized the ET-1-induced constriction of EPA than that of IPA. The combination of BQ-123 and BQ-788 increased the antagonistic response to ET-1 in IPA but not in EPA. Then, large constriction remained in IPA and EPA. The receptor nonselective antagonist PD-145065 almost completely inhibited the ET-1-induced constriction of IPA and EPA. BQ-123 completely inhibited the ET-3-induced constriction of EPA; however, it partially suppressed that of IPA. The combination of BQ-123 and BQ-788 completely inhibited the ET-3-induced constriction of IPA. These results demonstrate that the ET-1-induced constrictions are mediated by both ET(A) and ET(B) in IPA and by ET(A) in EPA. Because of differences in sensitivity to ET receptor antagonists for ET-1- and ET-3-induced constrictions, pharmacological heterogeneity of ET(A) is suggested. Additionally, endothelial denudation affected the ET-3-induced constriction of EPA, but not of IPA, and it didn't affect the response to ET-1. This suggests that the vasodilatory effect of endothelium on ET-3-induced vasoconstrictions varies depending on pulmonary vascular regions.

The effect of Cu2+ on rat pulmonary arterial rings.

In the current study, Cu2+ was tested for its ability to relax vessels and to accumulate cyclic GMP (cGMP) in rat pulmonary artery employing rat extrapulmonary arterial rings. Cu(2+)-induced relaxation was endothelium and concentration (in the range from 10(-7) to 10(-4) M) dependent. The content of cGMP in the rings was increased 1.7-fold with 10(-4) M Cu2+. NG-Monomethyl-L-arginine abolished both the copper-induced relaxation and the increase in cGMP of rings. Cu2+ and zaprinast, which inhibits phosphodiesterase activity, caused a synergistic increase in cGMP level in the rings, suggesting that Cu2+ enhanced cGMP level through a mechanism different from that of zaprinast, probably as a consequence of elevated accumulation of nitric oxide (NO). The magnitude of vasorelaxation observed due to simultaneous addition of Cu2+ and acetylcholine was additive, not synergistic. Cu2+ did not augment relaxation induced by exogenously added NO donor. These results suggest that Cu2+ elevates NO level in the rings not by prolonging the half-life of NO, but by activation of endothelial nitric oxide synthase and subsequently potentiating the action of NO on vascular tone.

Protective effects of BQ-123, an ETA receptor antagonist, against leukotoxin-induced injury in rat lungs.

We tested the hypothesis that BQ-123, a novel endothelin type A (ETA) receptor antagonist, protects the lung against leukotoxin 9,10-epoxy-12-octadecenoate (Lx), which causes acute lung injury in animals. In isolated rat lungs perfused with Earle's balanced salt solution, BQ-123 suppressed the Lx-induced increase in wet lung weight, wet lung weight/dry lung weight, the effluent perfusate lactic dehydrogenase activity, and effluent perfusate and lung tissue ET-1 levels. BQ-123 also significantly attenuated the Lx-induced increase of the pulmonary capillary filtration coefficient. Thus our experimental results indicate that the ETA receptor antagonist, BQ-123, protects against Lx-induced experimental lung vascular injury.

Effect of probucol, an oral hypocholesterolaemic agent, on acute tobacco smoke inhalation in rats.

1. We hypothesized that probucol, an oral hypocholesterolaemic agent, can suppress the oxidant stress induced by acute tobacco smoke inhalation in rats. We determined lung tissue glutathione (reduced and oxidized), lipid peroxide, tocopherol and plasma elastase inhibitory capacity, ferroxidase activity and lipid peroxide in rats after inhalation of tobacco smoke. 2. Rats treated with the probucol diet for 3 days or 4 weeks equally showed no suppression of plasma elastase inhibitory capacity and ferroxidase activity compared with control rats after acute tobacco smoke inhalation, although both animals treated with probucol for 3 days or 4 weeks had pharmacologically effective concentrations of probucol to lower plasma cholesterol but plasma cholesterol in rats treated with probucol for 3 days was still in the normal range. 3. Probucol treatment for 4 weeks lessened tobacco smoke-induced suppression of lung tissue glutathione, attenuated tobacco smoke-induced increases in lung tissue lipid peroxide and did not alter lung tissue tocopherol compared with control (lungs). 4. These findings demonstrate that probucol, via its antioxidant ability, confers a protective effect on lung exposed to acute tobacco smoke inhalation.

Adenine nucleotides via activation of ATP-sensitive K+ channels modulate hypoxic response in rat pulmonary artery.

We examined the role of ATP-sensitive K+ channels in hypoxic pulmonary vasoconstriction, using isolated rat pulmonary arterial rings. Isolated rat pulmonary arterial rings displayed a rapid contraction followed by relaxation under hypoxic conditions. The ATP-sensitive K+ channel blocker glibenclamide (concentration > 1 microM) or a hyperglycemic buffer (15 mM glucose) attenuated the hypoxic relaxation in a dose-dependent manner but did not affect the hypoxia-induced contraction. To examine the relationship between hypoxia, energy, and redox state, intracellular levels of adenine nucleotides and pyridine coenzymes were determined by high-performance liquid chromatography in freeze-dried isolated rat pulmonary arteries at three time points (0, 4, and 10 min) before and during hypoxia. Hypoxia time dependently decreased the ATP content and the ATP-to-ADP ratio and increased the ADP and the AMP content in association with a rapid increase in the NADH and the NADH-to-NAD+ ratio. Hyperglycemic buffer (15 mM glucose) suppressed the hypoxia-induced changes of the adenine nucleotides (the decrease of the ATP content and the ATP-to-ADP ratio) but did not affect the hypoxia-induced changes of the NADH and the NADH-to-NAD+ ratio. Hypoxia did not affect the NADP+ or the NADPH content of pulmonary arteries. These findings indicate that an ATP-sensitive K+ channel regulates the tone of rat pulmonary arteries. Furthermore, an imbalance of the energy state may be involved in ATP-sensitive K+ channel activation during hypoxic vasorelaxation.

Role of nitric oxide and superoxide anion in leukotoxin-, 9,10-epoxy-12-octadecenoate-induced mitochondrial dysfunction.

The present study was carried out to explore the involvement of nitric oxide (NO) and superoxide anion (O2.-) in Leukotoxin (Lx)-induced suppression of mitochondrial respiration. Glutamate- and succinate-dependent oxygen consumption and cytochrome c oxidase activity were assayed. Lx-induced mitochondrial damage was significantly attenuated by the pretreatment of lung with 4 x 10(-4) M NG-monomethyl-L-arginine (L-NMMA) or 500 units/ml superoxide dismutase (SOD) in ex vivo. However, L-NMMA plus SOD pretreatment showed no additive effect on the recovery of mitochondrial functions. The same assay was performed after the exposure of intact mitochondria to NO containing solution (1.25 x 10(-5) M) or 0.1 mM KO2/18-Crown-6 solution, which generated O2.-(6.4 x 10(-5) M). NO, but not O2.-, significantly inhibited the respiration of isolated mitochondria in vitro. Thus, there were great discrepancies in the involvement of NO and O2.- between ex vivo and in vitro system. Together with the previous reports, these facts suggested that the mechanisms by which NO and O2.- probably from vascular constituent cells inhibit mitochondrial respiration function of isolated perfused rat lung may not be simply due to their direct reactions with mitochondrial electron transport chain components, but may rely on the formation of peroxynitrite, and/or peroxynitrite-derived oxidants.

[The clinical study on secretory leukoprotease inhibitor (SLPI) in sera of patients with various pulmonary diseases].

It has been reported that secretory leukoprotease inhibitor (SLPI) can be a useful indicator for acute respiratory tract inflammation. In the present study, we attempted to measure automatically the serum concentration of SLPI by enzyme immunoassay (EIA) in patients with various pulmonary diseases. In this automatic measurement of SLPI, by which the results basically well-correlate with the manual method, we could measure many samples easily. Serum levels of SLPI in patients with various pulmonary diseases were significantly higher than those in healthy controls (50.5 +/- 9.8ng/ml). The serum concentration of SLPI in patients with inflammatory lung diseases correlated with that of C-reactive protein (CRP) or interleukin 6 (IL-6) significantly but not strongly. These results suggest that SLPI may be a useful indicator for local inflammation in respiratory tract. The serum concentration of SLPI in patients with lung cancer (71.1 +/- 10.8ng/ml), in particular adenocarcinoma, was significantly higher than that in healthy controls, but not correlated with other inflammatory markers.

Endothelin-1 potentiates leukotoxin-induced edematous lung injury.

We tested the hypothesis that leukotoxin (Lx), a cytochrome P-450-dependent linoleate product of leukocytes, can stimulate the release of endothelin-1 (ET-1) from the lung and further that exogenous ET-1 synergizes with Lx to produce edematous lung injury. In isolated rat lungs perfused with Earle's balanced salt solution, Lx (10 mumol) alone caused lung edema and increased the perfusate and lung tissue ET-1 levels. The combination of ET-1 (5 nM) and Lx (5 mumol), at concentrations that by themselves did not increase wet lung weight, significantly increased wet lung weight, wet-to-dry lung weight ratio, as well as the lung effluent lactate dehydrogenase activity. Pretreatment with BQ-123 (5 x 10(-6) M), an endothelin A receptor antagonist that significantly attenuated the ET-1 (5 nM)-induced increase in pulmonary arterial pressure (Ppa) and pulmonary capillary pressure (Ppc), suppressed the edematous lung injury generated by the combination of ET-1 and Lx, suggesting that the edema-enhancing effect of ET-1 in Lx-treated lungs occurred through endothelin A receptor-dependent elevation of Ppa and Ppc. Elevation of the pulmonary venous pressure in Lx-treated lungs (13.5 cmH2O) mimicked the effect of ET-1 on Ppa and Ppc and produced a degree of lung edema that was comparable to that after combined ET-1 + Lx treatment but without increase in the perfusate lactate dehydrogenase. These data support the idea that ET-1 and Lx promote lung edema in a synergistic fashion.

Leukotoxin, 9,10-epoxy-12-octadecenoate inhibits mitochondrial respiration of isolated perfused rat lung.

To investigate how mitochondrial function was affected in leukotoxin (Lx)-,9,10-epoxy-12-octadecenoate-induced lung injury, lung mitochondria were extracted from isolated perfused rat lung with or without Lx-induced edematous injury. In the lung treated with 30 mumol of Lx, the mitochondrial respiration rate in states 3 and 4 significantly decreased (without mitochondrial uncoupling) concomitantly with increased release of lactate dehydrogenase (LDH), a parameter for cellular damage, into the perfusate and decreased ATP content in the lung tissue compared with those of untreated lung. Moreover, 30 mumol of Lx resulted in significant inhibition of cytochrome-c oxidase activity (vs. vehicle control). In contrast, lower doses of Lx (10 mumol) caused lung edema and cellular damage without evidence for mitochondrial dysfunction. We also examined cellular and mitochondrial damage in hydrostatic lung edema. Such edema showed neither suppressed mitochondrial respiration nor elevated LDH activity in perfusate, although lung wet weight increased as much as it did after 30 mumol Lx treatment. Our results suggest that the ex vivo mitochondrial dysfunction is one of the secondary (vs. initial augmented permeability) but specific manifestations of toxicity of Lx, and together with the previous reports, the ex vivo damaging effect of Lx against mitochondria may be ascribed not to its direct action on mitochondria but to Lx-derived cellular mechanism(s).

Effects of insulin, insulin-like growth factor-I, and phorbol esters on neutral cholesteryl esterase activity in cultured rat vascular smooth muscle cells.

We investigated the effects of insulin, insulin-like growth factor-I (IGF-I), and phorbol 12-myristate 13-acetate (PMA) on neutral cholesteryl esterase activity in cultured rat vascular smooth muscle cells. Insulin and IGF-I at concentrations between 10(-9) mol/L and 10(-6) mol/L significantly decreased neutral cholesteryl esterase activity in growth-arrested vascular smooth muscle cells in a dose-dependent manner but with no influences on the intracellular concentration of 3',5'-adenosine monophosphate (cyclic AMP). Treatment of cells with KT5720 (10(-7) mol/L to 10(-5) mol/L), a specific inhibitor of cyclic AMP-dependent protein kinase, significantly decreased neutral cholesteryl esterase activity in a dose-dependent manner. Incubation of cells for 6 to 12 hours with PMA (10(-9) mol/L to 10(-6) mol/L), an activator of protein kinase C, significantly increased neutral cholesteryl esterase activity in a dose-dependent manner. However, down-regulation of protein kinase C activity by long-term incubation (18 to 48 hours) with PMA resulted in a significant decrease in neutral cholesteryl esterase activity. Treatment of cells with UCN-01 (10(-7) mol/L to 10(-5) mol/L), a specific protein kinase C inhibitor, decreased the enzyme activity in a dose-dependent manner and completely blocked the activation of the enzyme by PMA. When insulin or IGF-I at a concentration of 10(-6) mol/L was present in the medium containing CL 277,082--an inhibitor of acyl coenzyme A:cholesterol acyltransferase--cellular cholesteryl ester content of the cells significantly increased. In contrast, after the treatment with PMA at a concentration of 10(-6) mol/L in the presence of CL 277,082, the net cholesteryl ester content of the cells significantly declined. These data suggest that both insulin and IGF-I may increase cholesteryl ester accumulation in arterial smooth muscle cells by decreasing arterial cholesteryl ester hydrolysis. The data also suggest that neutral cholesteryl esterase is activated not only by cyclic AMP-dependent protein kinase but also by protein kinase C. Thus growth factors may exert their antilipolytic or lipolytic actions specifically by modulating neutral cholesteryl esterase activity in vascular smooth muscle cells. Neutral cholesteryl esterase of vascular smooth muscle cells may be regulated by recholesteryl esterase of vascular smooth muscle cells may be regulated by reversible phosphorylation, with the phosphorylated form being the active form.

Leukotoxin, 9,10-epoxy-12-octadecenoate causes edematous lung injury via activation of vascular nitric oxide synthase.

We examined the mechanism of leukotoxin, 9,10-epoxy-12-octadecenoate (Lx)-induced lung injury in blood-free, physiological salt solution-perfused rat lungs under constant flow conditions. Mean pulmonary arterial (Ppa) and pulmonary capillary pressure (Pcap, estimated by the double-occlusion method), wet lung weight (WLW), pulmonary capillary filtration coefficient (Kfc), lung perfusate lactate dehydrogenase (LDH) activity, and nitrite levels were assessed. Bolus injection of Lx (200 microM) caused insidious and significant lung weight gain, which was not associated with remarkable elevation of Ppa or Pcap but was associated with an increase of perfusate LDH activity and nitrite levels. Lx (20 microM) elevated Kfc, indicating that Lx had affected pulmonary vascular permeability. Because Lx causes endothelium dependent pulmonary vasodilation, we studied the effect of NG-monomethyl-L-arginine (L-NMMA), NG-monomethyl-D-arginine (D-NMMA), superoxide dismutase (SOD), human oxyhemoglobin (oxyHb), and methylene blue (MB) on Lx-induced lung injury. L-NMMA, SOD, and oxyHb, but not MB or D-NMMA, protected the lungs against Lx (200 microM)-induced injury. Lx increased pulmonary vascular permeability and caused lung injury. Because both nitric oxide synthase inhibitors and SOD inhibited the Lx-induced lung injury, it is possible that peroxynitrite is involved in the mechanism whereby Lx causes lung injury.

Enhancement of the binding of triglyceride-rich lipoproteins to the very low density lipoprotein receptor by apolipoprotein E and lipoprotein lipase.

The low-density lipoprotein (LDL) receptor plays a crucial role in cholesterol metabolism. A related protein, designated the very low density lipoprotein (VLDL) receptor, that specifically binds apolipoprotein (apo) E has recently been characterized and shown to be expressed in heart, muscle and adipose tissue and the human monocyte-macrophage cell line THP-1. The VLDL receptor binds and internalizes VLDL and intermediate density lipoprotein from Watanabe heritable hyperlipidemic (WHHL) rabbits as well as beta-migrating VLDL from cholesterol-fed rabbits but not LDL from WHHL rabbits. Chinese hamster ovary (CHO) cells transfected with the rabbit VLDL receptor cDNA have now been shown to bind or internalize VLDL (d < 1.006 g/ml) isolated from fasted normolipidemic human subjects with lower affinity than WHHL-VLDL or rabbit beta-VLDL. However, binding and internalization were markedly enhanced when fasted human VLDL was preincubated with either recombinant human apoE (3/3) or lipoprotein lipase (LPL) in CHO cells overexpressing the rabbit or human VLDL receptor. CHO cells transfected with both the rabbit VLDL receptor cDNA and the human LPL cDNA effectively bound, internalized, and degraded fasted human VLDL without pretreatment. Treatment of heparinase reduced the effect of LPL-mediated binding at 4 degrees C, but the inhibitory effect was lower at 37 degrees C. Pseudomonas LPL also enhanced the binding of human fasted VLDL to the VLDL receptor at 37 degrees C in CHO cells overexpressing the human VLDL receptor. Taken together, LPL causes the enhancement of triglyceride-rich lipoproteins binding to the VLDL receptor via both the formation of bridge between lipoproteins and heparan sulfate proteoglycans and its lipolytic effect. Ligand blot analysis showed that the apparent molecular mass of the VLDL receptor is 118 kDa, which is smaller than that of the LDL receptor. These results indicate that the VLDL receptor recognizes both triglyceride-rich lipoproteins that are also relatively rich in apoE, as well as the remnants of triglyceride-rich lipoproteins after catabolism and the interaction with heparan sulfate proteoglycans by LPL. The VLDL receptor may thus function as a receptor for remnants of triglyceride-rich lipoproteins in extrahepatic tissues.

Increased nitric oxide biosynthesis in leukotoxin,9,10-epoxy-12-octadecenoate injured lung.

We measured effluent nitric oxide levels using a chemiluminescence method from leukotoxin (Lx, a linoleate epoxide) injured isolated rat lungs perfused with physiological salt solution. Nitric oxide production from Lx-injured lung promptly increased and lasted for 20 min. Pretreatment with NG-monomethyl-L-arginine (LNMMA) significantly suppressed Lx-induced production of nitric oxide. Effluent from control lungs showed trace levels of nitric oxide. The wet to dry lung weight (WLW/DLW) after termination of the experiments was significantly elevated in Lx-treated lungs compared with that of LNMMA pretreated lungs or control lungs. There was a correlation between nitric oxide levels (at 10 min) and lung edema (WLW/DLW). Thus, nitric oxide plays a role in the pathogenesis of Lx-induced lung injury.

Autocrine production of endothelin-1 participates in the glucocorticoid-induced Ca2+ influx into vascular smooth muscle cells.

We determined whether endothelin-1 (ET-1) is associated with glucocorticoid-induced Ca2+ influx into vascular smooth muscle cells by examining the effects of the ETA receptor antagonist FR139317 on dexamethasone-induced 45Ca2+ uptake and dihydropyridine binding by rat A7r5 cells. FR139317 inhibited the dexamethasone-induced 45Ca2+ uptake and [methyl-3H]PN 200-110 binding in a dose-dependent manner. Slot blot analysis revealed that dexamethasone increased protein kinase C-alpha in A7r5 cells and that this effect was also abolished by FR139317. Dexamethasone stimulated the release of immunoreactive endothelin-1 from A7r5 cells into the culture medium. These results suggest that endothelin participates in the glucocorticoid-induced Ca2+ influx through dihydropyridine-sensitive channels in an autocrine manner, possibly linked to the activation of protein kinase C-alpha.

Lipid accumulation and foam cell formation in Chinese hamster ovary cells overexpressing very low density lipoprotein receptor.

The rabbit very low density lipoprotein receptor gene was introduced into LDL receptor-negative Chinese hamster ovary cells (ldl-A7). Incubation of the transfected cells with rabbit beta-VLDL (5 to 8 micrograms protein/ml), for 6 days, induced foam cell formation. The cells accumulated lipid droplets visualized by oil red-O staining; the cellular cholesteryl ester and triglyceride content increased two- to threefold. [3H]Oleate incorporation into cholesteryl [3H]oleate was stimulated threefold by incubation of cells with beta-VLDL (20 micrograms protein/ml). Northern blot analysis revealed the presence of VLDL receptor mRNA in rabbit resident alveolar macrophages. Incubation of the macrophages with beta-VLDL (20 micrograms protein/ml) for 24 hours induced foam cell formation but had virtually no effect on VLDL receptor mRNA abundance. These results suggest that the VLDL receptor on macrophages may play an important role in foam cell formation.

Relation of angiographically defined coronary artery disease and plasma concentrations of insulin, lipid, and apolipoprotein in normolipidemic subjects with varying degrees of glucose tolerance.

We investigated the association between hyperinsulinemia and changes in lipid, lipoprotein, and apolipoprotein that would increase the risk of coronary artery disease (CAD) independent of glucose tolerance. A coronary angiogram was recorded in 127 male subjects, including 41 with normal glucose tolerance, 41 with impaired glucose tolerance, and 45 with non-insulin-dependent diabetes mellitus (NIDDM). Subjects were divided into 2 groups according to results: the group with CAD (n = 94) and the group with normal coronary arteries (n = 33). All subjects were normolipidemic (total cholesterol < 230 mg/dl and triglycerides < 150 mg/dl). The CAD group had a significantly lower plasma level of high-density lipoprotein (HDL) cholesterol and apolipoprotein A-I (apo A-I) and a higher level of apolipoprotein B (apo B) than the normal group with normal glucose tolerance. In considering subjects with impaired glucose tolerance or NIDDM, the CAD group had a significantly lower plasma level of HDL cholesterol and apo A-I and a significantly higher plasma level of total cholesterol, triglycerides, and apo B than the normal group. In each of the subjects with normal and impaired glucose tolerance, and NIDDM, the elevation of plasma insulin concentration during both the complete test period and the early phase of an oral glucose challenge was significantly higher in the CAD than in the normal group. In all subjects, graded reductions in HDL cholesterol and apo A-I and graded increases in plasma total cholesterol, triglycerides, and apo B were observed with increasing tertiles of the postglucose challenge measurements of insulinemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Diltiazem inhibits DNA synthesis and Ca2+ uptake induced by insulin, IGF-I, and PDGF in vascular smooth muscle cells.

Proliferation of vascular smooth muscle cells (VSMC) has been shown to play a key role in the atherosclerotic lesions. It has been demonstrated that serum-derived peptidic growth factors, such as insulin, platelet-derived growth factor (PDGF), or epidermal growth factor (EGF), provide mitogenic signals in VSMC and that the interplay of Ca2+ and other messengers is necessary for triggering proliferation. Since Ca2+ channel blockers act on the voltage-dependent Ca2+ channel to inhibit Ca2+ influx, it is conceivable that they affect the proliferative action of growth factors. In this study we have evaluated the effects of diltiazem, a 1,5-benzothiazepine-derived Ca2+ channel blocker, on [3H]thymidine incorporation into DNA stimulated by insulin, insulinlike growth factor I (IGF-I), or PDGF in cultured VSMC from rat aorta. We have also investigated the effects of insulin, IGF-I, and PDGF on Ca2+ uptake in VSMC. After exposure to insulin (10(-10) to 8 x 10(-6) M) or IGF-I (10(-10) to 10(-7) M) for 48 hours, VSMC incorporated [3H]thymidine to 200-280% of maximum (with insulin or IGF-I alone) compared to control. The effect of IGF-I was approximately 10-100 times more potent than that of insulin. PDGF (0.5-15 ng/ml) also induced an increase in [3H]thymidine incorporation into DNA of VSMC. Additivity is observed between PDGF with insulin or IGF-I, but not between insulin and IGF-I. Sixty minute treatment with insulin (5 x 10(-8) to 10(-6) M), IGF-I (10(-8) to 10(-6) M), or PDGF (1.0-15.0 ng/ml) increased the unidirectional 45Ca2+ uptake during a 5 minute period.(ABSTRACT TRUNCATED AT 250 WORDS)

Increase in neutral cholesteryl ester hydrolase activity produced by extralysosomal hydrolysis of high-density lipoprotein cholesteryl esters in rat hepatoma cells (H-35).

The metabolism of high-density lipoprotein-associated cholesteryl esters (HDL-CE) in liver cells is not well understood. We studied the possible role of lysosomal and extralysosomal pathways on such metabolism by measuring the uptake and hydrolysis of HDL-CE in H-35 rat hepatoma cells. Incubation of cells with [3H]cholesteryl ester-labeled HDL led to the intracellular accumulation of both 3H-free cholesterol and [3H]cholesteryl ester. The ratio of 3H-free cholesterol/[3H]cholesteryl ester increased with an increase in incubation time even in the presence of chloroquine. Because chloroquine did not inhibit the conversion of cholesteryl ester to free cholesterol, the hydrolysis of HDL-CE may have been catalyzed by an extralysosomal enzyme, perhaps by neutral cholesteryl ester hydrolase (NCEH). When we incubated cells with increasing concentrations of HDL, NCEH activity increased. This increase in enzyme activity was not inhibited by the addition of chloroquine. A complex of dimyristoylphosphatidylcholine (DMPC)/apo HDL/cholesteryl ester enhanced the activity as well as native HDL. Neither the DMPC/apo HDL nor the DMPC/cholesteryl ester complex affected the activity, suggesting that apo HDL may be required for the uptake of HDL-CE. The present study demonstrated that the extralysosomal hydrolysis by NCEH is operating in the metabolism of HDL-CE in hepatoma cells.