Biomedical subjects

T N Tulenko

Publications and source records attributed to T N Tulenko.

At least 19 recordsLinked to original sources

PubMed2003

Cardiac systolic and diastolic dysfunction after a cholesterol-rich diet.

BACKGROUND: Although hypercholesterolemia is a well-established risk factor for coronary artery disease, little is known regarding its direct effects on cardiac function. METHODS AND RESULTS: We examined the effects of cholesterol feeding (0.5%) on cardiac function in rabbits. After 10 weeks, both systolic shortening and diastolic relaxation rates were impaired without any change in aortic pressure or ventricular hypertrophy. However, sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA)-2 mRNA levels were reduced within 4 days after initiation of cholesterol feeding. After this effect, SERCA-2 protein and SERCA-mediated Ca uptake into sarcoplasmic reticulum vesicles were impaired, and the ratio of MHC-beta to MHC-alpha mRNA increased 5-fold. Suppression of the SERCA-2 message correlated temporally with enrichment of the cardiac sarcolemma with cholesterol. CONCLUSIONS: These data demonstrate that dietary hypercholesterolemia induces a "cholesterol cardiomyopathy" characterized by systolic and diastolic dysfunction. These alterations were independent of vascular disease and demonstrate a dietary link to cardiac dysfunction.

Animals↗

PubMed2001

The smooth muscle cell membrane during atherogenesis: a potential target for amlodipine in atheroprotection.

BACKGROUND: Atherosclerotic disease has been present in the human population apparently from the beginning of time. However, it has only been in the 20th century that improvements in the control of infectious diseases have allowed the average life span to increase to the point where atherosclerosis has been able to affect the general population. By the middle of the 20th century, atherosclerosis had reached epidemic levels, and it is currently pandemic and increasing worldwide. Despite its growing significance to health care, we still know relatively little about the cellular basis for plaque genesis in the vessel wall. Current thinking holds that atherosclerosis is caused by an unchecked chronic inflammatory process involving the cells of the arterial wall and their interaction with LDL and various inflammatory cells. Considerable evidence suggests that the principal insults underlying atherogenesis are serum dyslipidemias and oxidative stress mediated primarily by oxidized LDL. However, just how these insults alter the cell biology of vascular cells and lead to the atherosclerotic phenotype is still under intense investigation. Moreover, recent clinical trials have provided evidence that certain classes of drugs, including newer calcium channel blockers (CCBs), can remodel the arterial smooth muscle cell (SMC) membrane and inhibit the progression of atherosclerotic disease. METHODS: This review summarizes our current thinking on atherogenesis in the arterial SMC and considers recent developments regarding alterations in the SMC membrane during the very early period of atherogenesis. We also discuss how certain CCBs might operate to produce atheroprotection. RESULTS: The SMC membrane becomes enriched in unesterified cholesterol soon after the development of serum hypercholesterolemia. With excess membrane cholesterol, the membrane becomes thicker and develops distinct cholesterol domains. These alterations in the membrane increase the permeability of SMC to calcium and induce a variety of alterations in SMC function that contribute to cellular atherogenic processes during plaque genesis. Amlodipine, a third-generation CCB, markedly inhibits the progression of lesions. The explanation of this novel action may lie in the effects of this drug on various potential cellular targets. CONCLUSIONS: Evidence is accumulating that excess membrane cholesterol may contribute to the cellular defects responsible for the transformation of the SMC to the atherosclerotic phenotype. Amlodipine, which has membrane-remodeling properties, is emerging as an important atheroprotective drug.

Amlodipine↗

PubMed2000

Membrane cholesterol content modulates activation of volume-regulated anion current in bovine endothelial cells.

Activation of volume-regulated anion current (VRAC) plays a key role in the maintenance of cellular volume homeostasis. The mechanisms, however, that regulate VRAC activity are not fully understood. We have examined whether VRAC activation is modulated by the cholesterol content of the membrane bilayer. The cholesterol content of bovine aortic endothelial cells was increased by two independent methods: (a) exposure to a methyl-beta-cyclodextrin saturated with cholesterol, or (b) exposure to cholesterol-enriched lipid dispersions. Enrichment of bovine aortic endothelial cells with cholesterol resulted in a suppression of VRAC activation in response to a mild osmotic gradient, but not to a strong osmotic gradient. Depletion of membrane cholesterol by exposing the cells to methyl-beta-cyclodextrin not complexed with cholesterol resulted in an enhancement of VRAC activation when the cells were challenged with a mild osmotic gradient. VRAC activity in cells challenged with a strong osmotic gradient were unaffected by depletion of membrane cholesterol. These observations show that changes in membrane cholesterol content shift VRAC sensitivity to osmotic gradients. Changes in VRAC activation were not accompanied by changes in anion permeability ratios, indicating that channel selectivity was not affected by the changes in membrane cholesterol. This suggests that membrane cholesterol content affects the equilibrium between the closed and open states of VRAC channel rather than the basic pore properties of the channel. We hypothesize that changes in membrane cholesterol modulate VRAC activity by affecting the membrane deformation energy associated with channel opening.

Animals↗

PubMed1999

Expression of the heterogenous nuclear ribonucleoprotein complex K protein and the prolyl-4-hydroxylase alpha-subunit in atherosclerotic arterial smooth muscle cells.

Smooth muscle cells (SMC) play a major role in the formation of atherosclerotic lesions found on major blood vessels. SMC proliferation, migration, and protein synthesis promote the progression of the early lesion, the fatty streak, into a complex myointimal fibrous plaque. To investigate altered gene expression in SMC during atherogenesis, we characterized differences between SMC from normal rabbits, rabbits fed a 2% cholesterol diet, and Watanabe Heritable Hyperlipidemic rabbits (WHHL). We detected and isolated a 501 bp cDNA fragment representing the A isoform of heterogenous nuclear ribonucleoprotein complex K (hnRNP-K) and a 281 bp cDNA fragment representing the prolyl-4-hydroxylase alpha-subunit (alphaPH) mRNAs. hn-RNP-K was upregulated in SMC from cholesterol-fed rabbits isolated in primary culture, as well as in SMC medial tissue from both the cholesterol-fed and WHHL rabbits. alphaPH was upregulated in SMC from the cholesterol-fed rabbits isolated in primary culture and in the tissue from WHHL rabbits. These data demonstrate genes consistent with increased proliferation and collagen production are upregulated in SMC during atherogenesis and may shed new light on gene expression changes and corresponding phenotype changes in SMC during atherogenesis.

Animals↗

PubMed1999

Rapid and opposite effects of cortisol and estradiol on human erythrocyte Na+,K+-ATPase activity: relationship to steroid intercalation into the cell membrane.

We determined whether two naturally occurring steroids, cortisol and 17beta-estradiol (E2), can rapidly modulate the activity of an important membrane protein, human erythrocyte (RBC) Na+,K+-ATPase, an enzyme that does not bind either hormone directly. We also determined the membrane binding locations for cortisol and E2 and their effects on membrane molecular structure and fluidity. Direct application of both steroids to intact human RBC significantly altered maximum ouabain-sensitive 86Rb uptake within 5 min: Cortisol decreased it by 24%, whereas E2 increased it by 18%. As determined by small angle x-ray diffraction, these steroids occupied distinct time-averaged binding locations in the RBC membrane, cortisol localizing near the bilayer surface, 14-29 A from the bilayer center, and E2 localizing deep within the hydrocarbon core, 0-7 A from the bilayer center. Neither steroid significantly changed overall bilayer width or membrane fluidity. These data suggest that cell membrane protein function can be altered rapidly and differentially by naturally occurring steroids. This effect did not appear to be related to the different binding locations of the steroids in the membrane or to their influence on membrane fluidity.

Adult↗

PubMed1999

Atheroprotection with amlodipine: cells to lesions and the PREVENT trial. Prospective Randomized Evaluation of the Vascular Effects of Norvasc Trial.

Oxidized lipid and calcium regulatory abnormalities appear to play important roles in early atherogenesis secondary to cholesterol enrichment of the cell membrane in endothelial and arterial smooth muscle cells (SMCs). However, the link between the two is poorly understood. The findings reviewed here demonstrate that amlodipine has membrane-modifying and antioxidant actions at the cell membrane level in addition to its classical calcium channel blocking properties. These multiple pharmacologic actions may explain the cellular mechanisms of the atheroprotective effects of amlodipine in spontaneous atherogenesis and in accelerated atherosclerotic syndromes. Recent animal model studies have demonstrated that amlodipine inhibits the progression of atherosclerotic lesions and protects against restenosis after angioplasty. Amlodipine inhibits the cholesterol-induced increase in calcium permeability in SMCs, and has been shown to repair abnormalities in SMC membrane structure. Recent data have also demonstrated that amlodipine has a marked antioxidant action in membrane bilayers enriched with polyunsaturated fatty acids. However, these findings have been in animal models only; the efficacy of amlodipine in atheroprotection in humans cannot be predicted. The PREVENT trial has therefore been launched to examine the atheroprotective potential of amlodipine in spontaneous lesion development in humans with ischemic heart disease and in the prevention of restenosis after angioplasty.

Amlodipine↗

PubMed1999

Sex differences in African-Americans regarding sensitivity to insulin's glucoregulatory and antilipolytic actions.

OBJECTIVE: The purpose of this study was to determine if there are sex differences in African-Americans regarding the effect of obesity on sensitivity to insulin as a glucoregulatory and antilipolytic hormone. RESEARCH DESIGN AND METHODS: Data from study participants, 127 nondiabetic African-Americans (mean age 32 +/- 4 years), included anthropometric measurements, an oral glucose tolerance test (OGTT), a 2-h euglycemic-hyperinsulinemic clamp, and a fasting triglyceride level. Sensitivity to insulin as a glucoregulatory hormone was determined by M/FFM, where M is the mean glucose infusion rate during the second hour of the clamp and FFM is fat-free mass. Sensitivity to insulin's antilipolytic action was assessed during the OGTT by the percent suppression of free fatty acid (FFA) concentrations between 0 and 120 min. The higher the suppression of FFAs, the greater the sensitivity to insulin's antilipolytic action. RESULTS: The participants were classified by BMI into three groups: nonobese (31 men, 24 women), obese (17 men, 14 women), and severely obese (12 men, 29 women). The women had higher percentages of body fat (P < 0.001), and the men had greater FFM (P < 0.001). The M/FFM values for men versus women in each BMI group were nonobese, 8.8 +/- 2.8 vs. 10.8 +/- 4.4; obese, 7.2 +/- 3.4 vs. 8.5 +/- 3.4; and severely obese, 4.7 +/- 2.1 vs. 6.1 +/- 2.2. The difference between the BMI groups was significant (P < 0.001), as was the difference between men and women (P < 0.01). In addition, there was a significant sex difference in percent suppression of FFAS (P < 0.001). The men and women had similar fasting insulin and FFA concentrations; however, in the men only, the percent suppression of FFA declined with increasing obesity (nonobese, 83 +/- 15%; obese, 73 +/- 18%; and severely obese, 69 +/- 19%; P = 0.02). The women in all three BMI groups had lower FFA levels of 86-88%. CONCLUSIONS: Obese African-American men and women are resistant to insulin as a glucoregulatory hormone, but only obese men are resistant to insulin's antilipolytic action; obese African-American women are sensitive to insulin's antilipolytic action. The combined presence of sensitivity to insulin's antilipolytic action with resistance to insulin's glucoregulatory action in obese African-American women may contribute to their high prevalence of obesity and type 2 diabetes.

Adult↗

PubMed1998

Antioxidant and cytoprotective activities of the calcium channel blocker mibefradil.

Mibefradil is a new calcium channel antagonist (CCA) that acts on both L- and T-type channels, with 10-fold selectivity for T-type channels. In this study, the structural interactions of mibefradil with cardiac membrane lipid bilayers were directly examined with small-angle x-ray diffraction approaches and correlated with lipid peroxidation and bovine aortic endothelial cell viability assays. Electron density profiles (A vs electrons/A3) calculated from the diffraction data (37 degrees C) demonstrated that mibefradil had an equilibrium location in the hydrocarbon core/headgroup region of the cardiac bilayer, 12-27 A from the center of the membrane. Mibefradil also effected a pronounced reduction in electron density 0-11 A from the center of the cardiac membrane concomitant with a 7.5% (3 A) decrease in membrane hydrocarbon core thickness; these changes in membrane structure were not observed with the phenylalkylamine verapamil, a CCA with some structural similarity to mibefradil. As a result of membrane physico-chemical interactions, mibefradil inhibited (10-500 nM) lipid peroxide formation in liposomes enriched in polyunsaturated fatty acids. In aortic endothelial cells, mibefradil also inhibited loss of cell viability (IC50 of 2 microM) following acute oxy-radical generation by dihydroxyfumarate and Fe-ADP; the order of potency was mibefradil > verapamil > diltiazem. These findings indicate that the chemical structure of mibefradil contributes to biophysical interactions with the cell membrane that underlie antioxidant and cytoprotective activities in models of oxidative stress.

Animals↗

PubMed1998

Thrombospondin-1 is elevated with both intimal hyperplasia and hypercholesterolemia.

BACKGROUND: Thrombospondin-1 (TSP-1) is important in platelet adhesion and aggregation, inflammation, cell to cell interaction, angiogenesis, and smooth muscle cell (SMC) proliferation. TSP-1 expression increases rapidly with injury. Therefore, we hypothesize that TSP-1 may play a role in the development of intimal hyperplasia (IH). The purpose of this study is to examine the interaction between cholesterol and TSP-1 on SMC proliferation and to quantitatively assess TSP-1 expression in an established model of IH, with and without underlying cholesterol-induced atherosclerosis. MATERIALS AND METHODS: In vitro, rabbit aortic SMC culture studies were performed to see the effect of TSP-1 antibodies on PDGF and, separately, cholesterol-induced SMC proliferation. In vivo, 23 rabbits were fed either a regular or a high-cholesterol diet. Hypercholesterolemia was confirmed by measurement of serum levels. Subsets underwent intraluminal aortic injury. Aortas were harvested 8-10 weeks later. Arterial wall TSP-1 was evaluated immunohistochemically and quantified by computer image analysis. RESULTS: In vitro, TSP-1 antibodies were able to inhibit PDGF and cholesterol-induced SMC proliferation (P < 0.05). In vivo, TSP-1 was found predominantly in the extracellular matrix in the rabbit aorta. IH was uniformly seen status-post angioplasty. Hyperplasia was more prominent in samples from hypercholesterolemic animals. ANOVA and Student's t test analyses demonstrated significantly more TSP-1 in the high-cholesterol/angioplasty group than in all other groups (P = 0.0006 vs regular diet/no angioplasty group). CONCLUSIONS: These data are consistent with the hypothesis that TSP-1 contributes to the development of IH. This study suggests that injured arteries in hypercholesterolemic atherosclerotic rabbits overexpress TSP-1.

Animals↗

PubMed1998

Physical effects of cholesterol on arterial smooth muscle membranes: evidence of immiscible cholesterol domains and alterations in bilayer width during atherogenesis.

Small angle X-ray diffraction was used to examine arterial smooth muscle cell (SMC) plasma membranes isolated from control and cholesterol-fed (2%) atherosclerotic rabbits. A microsomal membrane enriched with plasma membrane obtained from animals fed cholesterol for up to 13 weeks showed a progressive elevation in the membrane unesterified (free) cholesterol:phospholipid (C/PL) mole ratio. Beyond 9 weeks of cholesterol feeding, X-ray diffraction patterns demonstrated a lateral immiscible cholesterol domain at 37 degrees C with a unit cell periodicity of 34 A coexisting within the liquid crystalline lipid bilayer. On warming, the immiscible cholesterol domain disappeared, and on cooling it reappeared, indicating that the immiscible cholesterol domain was fully reversible. These effects were reproduced in a model C/PL binary lipid system. In rabbits fed cholesterol for less than 9 weeks, lesser increases in membrane C/PL mole ratio were observed. X-ray diffraction analysis demonstrated an increase in membrane bilayer width that correlated with the C/PL mole ratio. This effect was also reproduced in a C/PL binary lipid system. Taken together, these findings demonstrate that in vivo, feeding of cholesterol causes cholesterol-phospholipid interactions in the membrane bilayer that alter bilayer structure and organization. This interaction results in an increase in bilayer width peaking at a saturating membrane cholesterol concentration, beyond which lateral phase separation occurs resulting in the formation of separate cholesterol bilayer domains. These alterations in structure and organization in SMC plasma membranes may have significance in phenotypic modulation or aortic SMC during early atherogenesis.

Animals↗

PubMed1997

Cholesterol, calcium and atherosclerosis: is there a role for calcium channel blockers in atheroprotection?

It is well known that the atherogenic dyslipidemias of either elevated serum LDL or reduced HDL levels correlate with the degree and severity of atherosclerosis. However, how this leads to atherogenesis is poorly understood. A role for cellular oxidative stress mediated by oxidized LDL has gained widespread acceptance, but this pathway is unlikely to be the sole atherogenic signal. Recent evidence obtained from arterial smooth muscle cells (SMC) and endothelial cells (EC) is consistent with another pathway that may explain, in part, the early alterations contributing to the initiation of cellular atherogenic modifications. This pathway involves enrichment of the cell plasma membrane with cholesterol. In SMC, in vitro (cell culture) and in vivo (cholesterol feeding) experiments demonstrate that cholesterol enrichment of the SMC membrane occurs rapidly and is associated with an increase in membrane bilayer width, calcium permeability, and cell proliferation. Removal of excess membrane cholesterol with human HDL restores these alterations, suggesting that this membrane structural 'defect' mediates these changes in cell function. In vitro, the increased calcium permeability is inhibitable by calcium channel blockers (CCBs), but in vivo, a calcium 'leak' pathway develops that is virtually uninhibitable. It is not surprising that the literature on the application of CCBs for atheroprotection is not wholly convincing. However, with the advent of the new third generation of CCBs, new hope arises. One of the first CCBs of this generation is amlodipine (Norvasc), a charged dihydropyridine that has a remarkable pharmacologic profile. First, it is markedly lipophilic allowing it to partition readily into cell membranes. Second, in the membrane it has the ability to re-order, or restore, the 'swollen' membrane bilayer back to normal in atherosclerotic SMC. Third, it has potent antioxidant properties. Fourth, it appears to inhibit the expression of a variety of genes implicated in atherogenesis. Fifth, it is a CCB. Amlodipine has demonstrated atheroprotection in both rabbit and subhuman primate models of this disease. We propose that cellular alterations induced by enrichment of the cell membrane with cholesterol, which appears to modulate SMC to the atherosclerotic phenotype, are inhibitable by amlodipine through a combination of its varied pharmacologic properties. The potential for atheroprotection with amlodipine is currently being investigated in a human trial (PREVENT trial) and the results of this trial will determine the relevance of the preclinical findings to humans.

Amlodipine↗

PubMed1997

The relationship in African-Americans of sex differences in insulin-mediated suppression of nonesterified fatty acids to sex differences in fasting triglyceride levels.

Insulin is a potent antilipolytic hormone that promotes the deposition of fat and decreases the release of nonesterified fatty acids (NEFA) from adipose tissue. The purpose of this study was to investigate in African-Americans (AAs) sex differences in insulin-mediated suppression of plasma NEFA and fasting triglyceride (TG) levels. Ninety AAs, 44 men and 46 women with a mean age of 34 +/- 8 years were classified by body mass index (BMI) into three groups: non-obese (22 men and 18 women), obese (12 men and 10 women), and severely obese (10 men and 18 women). In each BMI group, women versus men had greater percent body fat (non-obese, 30 +/- 6 v 18 +/- 6, P < .001; obese, 36 +/- 3 v 26 +/- 2, P < .001; and severely obese, 39 +/- 4 v 29 +/- 4, P < .001). An oral glucose tolerance test (OGTT) was performed with fasting TG levels and plasma insulin and NEFA concentrations obtained at 0, 30, 60, and 120 minutes. In women, insulin-mediated NEFA suppression was similar in each of the three BMI groups (non-obese, 85% +/- 14%; obese, 88% +/- 11%; and severely obese, 87% +/- 10%; P = .8). In men, the percent suppression of NEFA declined with increasing obesity (non-obese, 83% +/- 14%; obese, 71% +/- 21%; and severely obese, 68% +/- 16%; P = .04). Changes in NEFA suppression were reflected in the fasting TG levels. TG levels in women were similar in each BMI group (non-obese, 71 +/- 39 mg/dL; obese; 69 +/- 21; severely obese, 79 +/- 30; P = .7). In contrast, fasting TG levels for men were higher in the higher BMI groups. Plasma TG levels in men were 87 +/- 41 mg/dL for obese, 113 +/- 65 for obese, and 169 +/- 81 for severely obese (P = .001). These data demonstrate sex differences in insulin-mediated NEFA metabolism. In AA women, the maintenance of sensitivity to insulin-mediated suppression of NEFA regardless of the degree of obesity may contribute to the normal plasma TG levels. For AA men, the resistance to insulin-mediated suppression of NEFA in the higher BMI categories may allow more NEFA to be released from adipose tissue into the circulation and available to the liver for synthesis into TG-containing lipoproteins.

Adult↗

PubMed1997

Interferon-induced growth arrest is mediated by membrane structural changes.

Interferon-gamma (IFN-gamma) is an immunomodulator shown to augment the expression of major histocompatibility (MHC) class I/class II antigens on the cell surface. In previous studies, we have demonstrated that the enhanced expression of these antigens on the cell surface is in part due to IFN-gamma-mediated abrogation of antigen shedding. In this study, we demonstrate that IFN-gamma induces structural changes in the cell membrane by altering the cholesterol/phospholipid ratio. Furthermore, such changes not only mediate enhanced expression of antigen on the cell surface but may drive the cells to growth arrest and apoptosis. These results were obtained by employing x-ray diffraction, electron microscopy, and DNA analysis.

Adjuvants, Immunologic↗

PubMed1995

Nickel is a specific inhibitor for the binding of activated alpha 2-macroglobulin to the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor.

The low density receptor-related protein/alpha 2-macroglobulin receptor (LRP/alpha 2-MR) binds to several ligands involved in lipoprotein and protease clearance. The receptor-associated protein (RAP) inhibits the binding of all known ligands. We studied the inhibition by Ni2+ of the binding of different ligands to cells and to the purified LRP/alpha 2-MR. Ni2+ inhibited all of the specific binding of radiolabeled methylamine-activated alpha 2-macroglobulin (125I-alpha 2-M*) to rabbit aortic smooth muscle cells (SMC), rat hepatoma Fu5AH, and mouse fibroblast L cells. Ni2+ also inhibited the binding of trypsin-activated alpha 2-macroglobulin to SMC but did not affect the binding of RAP, Pseudomonas exotoxin A, or low-density lipoproteins. The inhibition of alpha 2-M* binding by Ni2+ was not due to its interaction with alpha 2-M*. Preincubation of SMC with Ni2+ followed by ligand binding suggested that Ni2+ binds to cell-surface molecules and inhibits the binding of alpha 2-M* but does not affect RAP binding. Most of the binding of alpha 2-M* to SMC was due to its binding to the LRP/alpha 2-MR, as opposed to the recently described signaling receptor, as demonstrated by the inhibition of this binding by the RAP. Moreover, the inhibition of alpha 2-M* binding to the LRP/alpha 2-MR by Ni2+ was demonstrated using purified receptor immobilized on microtiter plates. Two to three molecules of 63Ni2+ bound to the immobilized receptor with equal affinity but not to alpha 2-M*. The specific binding of alpha 2-M* to the immobilized receptor was inhibited in the presence of nickel.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Atherosclerosis alters the composition, structure and function of arterial smooth muscle cell plasma membranes.

The object of this study was to examine changes in plasma membranes of arterial smooth muscle (ASM) during atherogenesis obtained from cholesterol-fed (2%) rabbits. A microsomal fraction highly enriched with plasma membrane markers was prepared by subcellular organelle fractionation from ASM freshly isolated from the thoracic aorta. The membranes were analyzed for unesterified (free) cholesterol (FC) content, membrane bilayer structural parameters (X-ray diffraction), phospholipid (PL) composition, and Na+/K(+)-ATPase activity and kinetics. Following 8 weeks on diet, membrane FC content increased 67.1%. Small angle X-ray diffraction demonstrated an increase in membrane hydrocarbon core electron density and an increase in overall lipid bilayer width (56-62 A). This increase in bilayer width was highly correlated with the membrane FC content (r = 0.992). Both membrane FC content And bilayer width independently correlated with time on cholesterol diet. The phospholipid profile of the membrane revealed a 16.4% increase in phosphatidylcholine (PC), 19.3% decrease in phosphatidylethanolamine (PE) and 62.8% increase in sphingomyelin (SM) content with no change in total PL content. Na+/K(+)-ATPase activity was decreased 52.2% (P < 0.005), and [3H]ouabain binding kinetics demonstrated a 27.6% decrease in maximum binding sites (Bmax) (P < 0.01) while the dissociation constant (Kd) remained unaltered. Membranes obtained from control ASM cells enriched with FC in culture demonstrated changes similar to those in atherosclerotic ASM membranes including an increase in membrane FC content, an increase in bilayer width, and a decrease in Na+/K(+)-ATPase activity with decreased ouabain Bmax. These data demonstrate marked compositional, structural and functional changes in ASM cell membrane characteristics in dietary atherosclerosis. These changes were highly correlated with cholesterol accumulation in the plasma membrane bilayer and were observed before the appearance of visible lesions. We suggest that these membrane defects may be linked with early atherogenesis.

Animals↗

PubMed1995

Altered contractile and ion channel function in rabbit portal vein with dietary atherosclerosis.

This study was performed to determine the effects of dietary atherosclerosis on the pharmacology and ion channel properties of rabbit portal vein (PV). New Zealand White rabbits were fed normal rabbit chow +/- 2% cholesterol for 10 wk. Contractions to norepinephrine (NE) and serotonin were studied under isometric conditions with longitudinal strips. Ca2+ and K+ currents (ICa and IK, respectively) were recorded in freshly dispersed myocytes by whole cell voltage clamp methods. Cholesterol feeding increased total plasma cholesterol levels from 28.4 +/- 5.2 to 1,387 +/- 172 mg/dl as well as the cholesterol-to-phospholipid molar ratio of the PV from 0.34 +/- 0.02 to 0.66 +/- 0.08. Only maximum contractile responses to serotonin were larger in atherosclerotic PV when normalized to the maximum KCl response. Concentration-active stress curves of the atherosclerotic PV to NE and serotonin were shifted to the left. Maximum values of ICa were larger in myocytes from atherosclerotic compared with control animals (4.4 +/- 0.4 vs. 3.1 +/- 0.2 pA/pF, P < 0.05). The voltage dependence of activation and availability of ICa was shifted toward more negative potentials by approximately 10 mV. Whole cell K+ currents were smaller in atherosclerotic myocytes. At a test voltage of +20 mV, IK averaged 14.9 +/- 2.8 pA/pF in control compared with 7.7 +/- 0.8 pA/pF in atherosclerotic myocytes from a holding potential of -80 mV with external Ca2+ concentration of 5 mM. The reversal potential for IK tail currents was significantly less negative in atherosclerotic myocytes (-70 +/- 1 vs. -64 +/- 1 mV).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Phorbol esters and norepinephrine destabilize alpha 1B-adrenergic receptor mRNA in vascular smooth muscle cells.

The mechanism by which norepinephrine (NE) down-regulates alpha 1B-adrenergic receptor (alpha-AR) mRNA was studied in rabbit aortic smooth muscle cells. NE, phorbol esters, and bradykinin each decreased alpha-AR mRNA levels by 70-80%. The protein kinase C inhibitor (+)-1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) abolished the effects of phorbol esters and NE and decreased basal mRNA levels by 52 +/- 3%. Neither ryanodine nor EGTA inhibited down-regulation of alpha-AR mRNA by NE. Actinomycin D caused alpha-AR mRNA level to decrease with a half-life of 3.2 +/- 0.4 h and blocked the effect of H-7 to decrease basal alpha-AR mRNA level. Both NE and phorbol esters increased the rate of alpha-AR mRNA degradation. In NE-desensitized cells, phorbol esters and bradykinin each caused the expected down-regulation of alpha-AR mRNA. The protein phosphatase inhibitor okadaic acid prolonged the normally transient effect of NE for at least 24 h. We conclude that protein kinase C exerts two opposing effects on alpha-AR mRNA levels, 1) a decrease in the stability of the mRNA that requires the sustained phosphorylation of a protein kinase C substrate and 2) a permissive effect on alpha-AR gene transcription.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1994

Increase in intracellular calcium ion in smooth muscle cells induced by low-density lipoprotein.

It was investigated whether low-density lipoprotein (LDL) had effects on intracellular Ca2+ concentration in the smooth muscle cell (SMC). LDL promoted SMC proliferation and increased intracellular Ca2+ by a two-phase pattern, an initial peak and a following plateau. Each phase was suppressed by treatment with ryanodine or extracellular Ca(2+)-free buffer. This increase in intracellular Ca2+ was also suppressed by anti-LDL receptor antibody. Moreover, inositol triphosphate (IP3) was elevated with short-term LDL treatment. Since SMC proliferation is the most important event in atherosclerosis and LDL is one of the main risk factors, it was concluded that LDL might trigger SMC proliferation by increasing IP3 and intracellular Ca2+ through LDL receptor.

Animals↗