Role of Ca2+ transporters and channels in the cardiac cell volume regulation.

Na+/K+ pump is one of key mechanisms to maintain cell volume. When it is inhibited, cells are at risk of swelling. However, in guinea pig ventricular myocytes, the cell area as an index of cell volume was almost constant during 90 min Na+/K+ pump blockade with 40 microM ouabain despite the marked membrane depolarization. In this study, involvements of Ca2+ transporters and channels in the cardiac cell volume regulation were proposed by conducting the computer simulation in parallel with the experimental validation.

The ATP-sensitive K+ channel.

A small conductance K+ channel, that is inactivated by ATP, was recently found in the inner membrane of rat liver mitochondria (Inoue et al., 1991). This finding clearly indicates that a variety of K+ channels, showing ATP-sensitivity, are widely distributed. ATP is an important compound in view of its participation in oxidative phosphorylation and as the source of high-energy phosphate for nearly every energy-requiring reaction in the cell. Therefore, it is easy to speculate that transducing the ATP concentration within a cell into an electrical signal is vital for most living cells. The opening of the ATP-sensitive K+ channel by a decrease in the ATP level shifts the membrane potential in a negative direction and in general depresses cell function. The closing of the channel by an increase in ATP depolarizes the membrane and enhances membrane excitability. It might be speculated that a sequence of amino acids common for the binding site of ATP is preserved and combined with different types of K+ channels, so that the gating with ATP is quite similar between different K+ channels, but the conductance properties are different. The large variability in the value of K1/2ATP in the same cells or between different tissues might be due to modulation of the reaction of ATP and the binding site. These ideas will be substantiated by clarifying the molecular structure of the ATP-sensitive K+ channel in the near future. The molecular mechanisms for the selective channel blockers, sulfonylureas, and for the K+ channel openers should also be clarified.

Partial purification and properties of phospholipase A2 from the starfish, Asterina pectinifera.

1. Phospholipase A2 (phosphatide 2-acyl-hydrolase, EC 3.1.1.4) activity was shown to occur in the supernatant fraction of a freshly prepared homogenate from the pyloric caecum of starfish (Asterina pectinifera). 2. The phospholipase A2 has been isolated and purified 130-fold by ultracentrifugation, ammonium sulfate precipitation and column chromatographic procedures. 3. The purified enzyme was stable to heat at low pH values and the optimal pH was observed at approximately 9.0. 4. The enzyme activity was activated by Ca2+ and sodium deoxycholate, and was inhibited by EDTA.

Stimulatory effects of lipoprotein(a) and low-density lipoprotein on human umbilical vein endothelial cell migration and proliferation are partially mediated by fibroblast growth factor-2.

We previously reported a transient increase in plasma lipoprotein(a) (Lp(a)) concentrations following acute myocardial infarction and surgical operations, and demonstrated Lp(a) accumulation in healing tissues. In the present study, the stimulatory effect of Lp(a) on migration and proliferation of human umbilical vein endothelial cells (HUVEC) was assessed by quantitative assay methods and compared it with that of LDL. Lp(a) stimulated both migration and proliferation of HUVEC in a dose-dependent manner and the stimulatory activities for migration and proliferation were two times higher than those of LDL in terms of moles of apoB. In addition, this stimulatory activity of Lp(a) was not affected by the difference of Lp(a) phenotype. Although each neutralizing antibody to hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF) and interleukin-1beta (IL-1beta) had no further effect on migration and proliferation of HUVEC treated with Lp(a), only antibody to fibroblast growth factor-2 (FGF-2) partially suppressed them. Moreover, pertussis toxin, which inhibits FGF-2-stimulated endothelial cell movement, also partially suppressed Lp(a)-induced HUVEC migration. FGF-2 concentrations in the medium of HUVEC treated with Lp(a) were constant in spite of the increase in FGF-2 mRNA levels in HUVEC. Taken together, it is suggest that Lp(a) stimulates HUVEC migration and proliferation, which is mediated, at least in part, by FGF-2 and may promote the angiogenesis during wound healing.

Purification and properties of cholesterol ester hydrolase from human aortic intima and media.

1. Cholesterol ester hydrolase of human aortic intima and media was isolated and purified about 650-fold with 10-15% recovery of the original activity by sequential precipitation with 35% acetone, gel filtration on Sephadex G-75 and DEAE-cellulose column chromatography. 2. Two pH optima of 4.5-5.0 and 7.0-7.5 were consistently observed for the partially purified cholesterol ester hydrolase of human aortic intima and media. 3. In the system used in the present study, the increasing concentration of emulsifiers, sodium taurocholate and phosphatidylcholine, inhibited the activity of the neutral enzymes but not on the acid enzymes. On the contrary, reaction products, cholesterol and oleic acid, were much more inhibitory on the acid enzymes than on the neutral ones. 4. Results of studies on the effect of presentation of substrate on the enzyme activity and on the difference between acid and neutral enzymes are also discussed.

Regulatory volume decrease of cardiac myocytes induced by beta-adrenergic activation of the Cl- channel in guinea pig.

A new method was developed to automatically measure the thickness of a single ventricular myocyte of guinea-pig heart. A fine marker was attached on the cell's upper surface and changes in its vertical position were measured by focusing it under the microscope. When the osmolarity of the bath solution was varied, the cell thickness reached a new steady level without any obvious regulatory volume change within the period of observation up to 15 min. The cell thickness was 7.8 +/- 0.2 microns (n = 94) in the control Tyrode solution and was varied to 130.4 +/- 3.1% (n = 10), 119.1 +/- 1.1% (n = 50), 87.2 +/- 1.9% (n = 9), and 75.6 +/- 3.2% (n = 5) of control at 50, 70, 130, and 200% osmolarity, respectively. The application of a Cl- channel blocker, 500 microM anthracene-9-carboxylic acid (9AC) did not modify these osmotic volume changes. We discovered that the application of isoprenaline induced a regulatory volume decrease (RVD) in cells inflated by hypotonic solutions. This isoprenaline-induced RVD was inhibited by antagonizing beta-adrenergic stimulation with acetylcholine. The isoprenaline-induced RVD was mimicked by the external application of 8-bromoadenosine 3':5'-cyclic monophosphate. The RVD was inhibited by blocking the cAMP-dependent Cl- channel (ICl, rAMP) with 9AC but was insensitive to 4,4'-diisothiocyanostilbene-2,2'-dissulphonate (DIDS). Taken together these data suggest an involvement of ICl, cAMP activation in the RVD. Whole cell voltage clamp experiments revealed activation of ICl, cAMP by isoprenaline under the comparable conditions. The cardiac cell volume may be regulated by the autonomic nervous activity.

Plasma Fas ligand, an inducer of apoptosis, and plasma soluble Fas, an inhibitor of apoptosis, in patients with chronic congestive heart failure.

OBJECTIVES: This study sought to examine plasma levels of soluble Fas/APO-1 receptor (sFas), an inhibitor of apoptosis, and soluble Fas ligand (sFas-L), an inducer of apoptosis, and their relation to each other and to other clinical variables, such as New York Heart Association functional class, tumor necrosis factor (TNF) and interleukin-6 (IL-6) in congestive heart failure (CHF). BACKGROUND: It has been recently reported that apoptotic cell death occurs in myocytes of dogs with CHF. Hypoxia is frequently seen in advanced CHF and can stimulate Fas/APO-1 receptors (Fas) to induce apoptosis in cultured myocytes. Fas and Fas ligand (Fas-L) are cell-surface proteins and representative apoptosis-signaling molecules. Fas on the cell membrane induces apoptosis when it binds Fas-L or sFas-L. However, plasma sFas, a molecule lacking the transmembrane domain of Fas, blocks apoptosis by inhibiting binding between Fas and Fas-L or sFas-L on the cell membrane. At present, it is unknown whether plasma sFas-L and plasma sFas increase in the presence of cardiac disease. METHODS: The study included 70 patients (mean [+/-SEM] age 65 +/- 2 years, range 21 to 93) with chronic CHF (coronary artery disease in 28, dilated cardiomyopathy in 27, valvular heart disease in 15) and 62 age- and gender-matched normal control subjects. Plasma levels of sFas, sFas-L, TNF-alpha and IL-6 were measured by enzyme-linked immunosorbent assays using monoclonal anti-human antibodies. RESULTS: There was no significant difference in sFas-L levels between normal subjects and patients in functional classes I to IV; however, sFas increased with severity of functional classification, independent of the underlying disease. sFas levels were significantly higher even in patients in functional class II than in normal subjects and those in functional class I, and were highest in patients in functional class IV (normal subjects; 2.2 +/- 0.1 ng/ml; functional class I: 2.2 +/- 0.2 ng/ml; functional class II: 3.1 +/- 0.2 ng/ml; functional class III: 3.9 +/- 0.3 ng/ml; functional class IV: 5.1 +/- 0.6 ng/ml). Plasma sFas levels were significantly higher in patients with elevated pulmonary artery wedge pressure and a decresed cardiac index than in those with values in the normal range. In patients in functional class IV, there was no significant difference in plasma sFas levels between the survivors and non-survivors during 6-month follow-up. However, plasma levels of sFas tended to decrease in nine patients with clinical improvement (baseline sFas: 5.2 +/- 0.8 ng/ml; 6-month sFas: 4.3 +/- 0.5 ng/ml, p = 0.07) but were similar in patients with no change in functional class. TNF-alpha and IL-6 were increased significantly only in patients in functional class IV, as previously reported, but were not related to sFas. CONCLUSIONS: We found elevated levels of plasma sFas and no increase in plasma sFas-L in human CHF. The increase in sFas may play an important role in the pathophysiologic mechanisms of CHF.

Intracellular Ca(2+) dynamics and sarcomere length in single ventricular myocytes.

The measurements of the sarcomere length in dissociated cardiac ventricular myocytes are discussed using mainly our own experimental data. The striation periodicity of these unloaded cells was found to be that which is to be expected of a myocyte free of the ultrastructural constraints imposed upon it by the normal syncytial matrix of the ventricular wall. The sarcomere length and [Ca(2+)] relationship was consistent as expected from the intact tissue, when it was measured soon after partial rupturing the cell membrane. Miniature fluctuations of individual sarcomere length were demonstrated during rest, which was augmented by the Ca(2+) overload. The [Ca(2+)] could be estimated from the measurements of sarcomere length during the positive staircase of contraction. The usefulness of the optical measurement of sarcomere pattern was indicated.

Expression of activin A in human keratinocytes at early stages of cultivation.

Activins are members of the TGF-beta superfamily and are classified into 3 types: activin A, which consists of a homodimer of betaA, activin B, which consists of a homodimer of betaB, and activin AB, which consists of a heterodimer of betaAbetaB. We studied the expression of activin mRNAs by RT-PCR in normal human epidermis, cultured keratinocytes, and DJM-1 cells (a squamous cell carcinoma line). We could detect only activin A mRNA (betaA) in normal human epidermis. In cultured keratinocytes and DJM-1 cells, activin betaA mRNA was observed at 4 h but not at 96 h after plating. Activin A activity was detected in the conditioned medium of DJM-1 cells within 48 h. In addition, although follistatin mRNA was not observed in human epidermis in situ, it was transiently expressed in cultured cells at 4 h after plating. These findings suggest that the expression of these molecules in keratinocytes is associated with cell proliferation. In an in vitro tissue injury model, activin A was observed at the wound edge, where cell migration and proliferation may be activated. In DJM-1 cells cultured for 92 h, betaA mRNA was observed 4 h after injury treatment. These findings suggest that activin A acts as a potent inducer of proliferation in vitro, at least in keratinocytes.

Studies on apolipoprotein(a) phenotypes. Part 1. Phenotype frequencies in a healthy Japanese population.

The frequency distribution for serum lipoprotein(a) (Lp(a)) concentrations in healthy Japanese was highly skewed, with a mean +/- S.D. of 14.6 +/- 13.6 mg/dl and a median of 11.0 mg/dl. The present study provides the first evidence on the frequencies of Lp(a) phenotypes and alleles in healthy Japanese subjects. There was a strong inverse relationship between the apparent molecular weights of apo(a) isoforms and plasma Lp(a) concentrations, as reported previously. However, because of the considerable overlap between the Lp(a) concentrations of the different phenotypes, it was impossible to predict Lp(a) concentration from Lp(a) phenotypes, or vice versa. The present results suggest that the distribution of Lp(a) concentrations, mean and median values and Lp(a) phenotype and allele frequencies in healthy Japanese are not significantly different from the results for Europeans, whereas they are significantly different from other Asian populations, i.e. Chinese, Indians and Malaysians.

Elastin-lipid interaction in the arterial wall. Part 2. In vitro binding of lipoprotein-lipids to arterial elastin and the inhibitory effect of high density lipoproteins on the process.

The mechanism of lipoprotein binding to arterial elastin, and the inhibitory effect of high density lipoprotein (HDL) on the in vitro complex formation between plasma low density lipoprotein (LDL) and elastin were studied. The binding of LDL-cholesterol, phospholipids and triacylglycerols to delipidated elastin increased progressively with time over 24 h of incubation. The results of a kinetic study on lipoprotein-cholesterol concentration in the incubation medium, suggesting that the ability to bind cholesterol to elastin decreases in the following order: very low density lipoprotein (VLDL), LDL, intermediate density lipoprotein (IDL) and HDL, and that the capacities to bind to a fixed amount of elastin decrease in the order: LDL, IDL, VLDL and HDL. When a definite amount of LDL was incubated with elastin in the presence of increasing concentrations of HDL, the binding of lipids to elastin progressively decreased. On the other hand, no release of cholesterol, bound to elastin during preincubation with LDL, could be detected in additional incubations with HDL, apoHDL or apoHDL-phospholipid complex.

Plasma lipids and apolipoproteins as discriminators for presence and severity of angiographically defined coronary artery disease.

The relationship of coronary artery disease to with plasma lipids and apolipoproteins was examined in 100 male patients aged 32-69 years undergoing coronary angiography for suspected myocardial infarction. Patients with angiographically defined coronary artery disease had significantly lower plasma levels of high density lipoprotein (HDL) cholesterol, apolipoproteins A-I and A-II, and significantly higher values of low density lipoprotein (LDL) cholesterol and apolipoprotein B than those in patients without coronary artery disease. The ratios derived from the measurements as LDL cholesterol/HDL cholesterol, apo B/apo A-I and apo B/apo A-II were highly significantly increased in the patients with coronary artery disease. Coronary score values, by which the severity of coronary artery disease was quantified, were not related to plasma levels of the HDL components, while they were positively correlated with those of the LDL components. These results suggest that, in single measurements, plasma levels of the HDL components, HDL-cholesterol and apo A-I, contribute strongly to the discrimination between patients with coronary artery disease and those without this disease, whereas the LDL components, LDL-cholesterol and apo B are more suitable parameters for the severity of the disease than are the HDL components. The ratios of LDL cholesterol/HDL cholesterol, apo B/apo A-I and apo B/apo A-II were powerful discriminators for either presence or severity of coronary artery disease.

Inhibitory effect of high density lipoprotein subfractions on the in vitro binding of low density lipoproteins to arterial elastin.

The inhibitory effects of high density lipoprotein (HDL) subfractions on the in vitro complex formation between plasma low density lipoprotein (LDL) and arterial elastin were studied. The inhibitory effects were significantly higher with HDL3 than HDL2, and with HDL-without E than HDL-with E. The inhibitory effect of a phospholipid complex with apoHDL3 was higher than that with apoHDL2. In contrast with the inhibitory effects, the binding abilities of HDL2 and HDL-with E to elastin were significantly higher than those of HDL3 and HDL-without E. These results suggest that the inhibitory effects of HDL subfractions are not due to competitive binding with arterial elastin.

Changes of serum apolipoprotein levels after oral administration of fat in human subjects.

Changes of serum apolipoprotein levels were studied every hour for 6 h after the administration of 55 g butter to 8 healthy male subjects. The mean serum apo A-IV level was significantly increased at 4 h (P less than 0.05) after fat ingestion compared to the mean initial level, although no significant changes of levels in other apolipoproteins (apo A-I, A-II, B, C-II, C-III, and E) were observed. The mean apo A-IV level in the triglyceride-rich lipoprotein (TRL) fraction (d less than 1.006) increased progressively over 6 h. In all 8 subjects, the time of peak concentration of apo A-IV in TRL fraction was delayed by 1-2 h compared to that in whole serum. On the other hand, the mean apo B-48 level in the fraction reached a peak at 4 h. These results raise the possibilities that some apo A-IV, newly synthesized or already existing in intestinal cells, may be directly secreted into the venous circulation and that apo A-IV and apo B-48 may distribute differently in different sizes of chylomicron. Alternatively, the amount of each apolipoprotein synthesized may depend upon the content of ingested fat. It is suggested that apo A-IV production by intestinal cells does not appear to be regulated by the rate of fat transport, and that apo A-IV does not play an important role in chylomicron formation compared to apo B-48.

Elastin--lipid interaction action in the arterial wall. Part 1. Extraction of elastin from human aortic intima.

Elastin was extracted from human aortic plaque and adjacent grossly normal intima by the following methods: (1) 0.1 N NaOH at 100 degrees C, (2) hot NaOH and 0.2 M EDTA, (3) 5 M guanidine--HCl and collagenase, (4) guanidine--collagenase and dithioerythritol--urea--sodium dodecyl sulfate, (5) guanidine--collagenase and EDTA, (6) 10% NaCl and collagenase, and (7) NaCl--collagenase and EDTA. All elastin samples contained small amounts of carbohydrate and hydroxyproline. The lipid content of non-plaque intimal elastin samples was small (2--3%), whereas it increased to 4--6% in plaque intima. The lipid composition of elastin preparations varied significantly with the extraction procedure. Elastin from plaque intima contained significantly more cholesterol (50--60%) and less triglyceride and phospholipid than elastin of non-plaque intima (30--50% cholesterol). The contents of free and esterified cholesterol were comparable in all preparations. The main phospholipid in all samples was sphingomyelin, which comprised between 50 and 80% of the total phospholipid. Compared with NaOH-purified elastin, the other elastin samples were characterized by an increased phosphatidyl--choline content, while they all contained an almost equal amount of phosphatidylethanolamine. In elastin samples from plaque intima, the polar amino acids were increased, whereas cross-linking amino acids were decreased. The polarity and hydroxyproline content of elastin samples were slightly decreased after treatment with EDTA or dithioerythritol--urea--sodium dodecyl sulfate.

Reduction of serum lipoprotein(a) levels in hyperlipidaemic patients with alpha-tocopheryl nicotinate.

The effect of low dose (600 mg/day) alpha-tocopheryl nicotinate on serum lipoprotein(a) (Lp(a] concentration was studied in 28 hyperlipidaemic patients. Serum lipids, lipoproteins and apolipoproteins, except for Lp(a), tended to increase after treatment. In particular, the changes in HDL-cholesterol and apo C-II levels were statistically significant. On the other hand, serum Lp(a) levels in all patients decreased significantly after 2 months of treatment. Furthermore, no difference between before and after treatment was observed in the group with initial Lp(a) levels less than 18 mg/dl, whereas Lp(a) concentrations decreased significantly after treatment in the group with levels greater than or equal to 18 mg/dl. The effects of probucol and alpha-tocopheryl nicotinate on serum Lp(a), total cholesterol and HDL-cholesterol were entirely different. Possible mechanisms of alpha-tocopheryl nicotinate on serum Lp(a) and lipoprotein metabolism are discussed.

Studies on apolipoprotein(a) phenotypes. Part 2. Phenotype frequencies and Lp(a) concentrations in different phenotypes in patients with angiographically defined coronary artery diseases.

In the present paper, we have evaluated serum Lp(a) concentrations, the frequencies of Lp(a) phenotypes and alleles and the association between the Lp(a) phenotypes and serum Lp(a) levels in 470 patients with angiographically defined coronary artery disease (CAD). Serum Lp(a) concentrations were significantly increased in proportion to the number of diseased vessels in the CAD patients. The frequencies of Lp(a) phenotypes in the CAD patients were significantly different from those in healthy subjects. In particular, the frequency of double-band phenotypes was higher in the CAD group. The frequencies of Lp(a) alleles in the CAD patients, however, were not significantly different from those in the healthy subjects. There was a strong inverse relationship between the apparent molecular weights of apo(a) isoforms and serum Lp(a) concentrations. Lp(a) levels in the CAD patients were higher than those in the healthy subjects with the same phenotype. The present results suggest that it is important to consider some posttranslational or environmental modifications and other factors, in addition to the genetic factor, when assessing contributions to plasma Lp(a) levels.

Lipoprotein(a) in nonhuman primates. Presence and characteristics of Lp(a) immunoreactive materials using anti-human Lp(a) serum.

Lipoprotein(a) (Lp(a] immunoreactive materials were examined in serum samples from 77 nonhuman primates of 24 species by Ouchterlony's double diffusion procedure and an enzyme-linked immunosorbent assay (ELISA) using rabbit antisera to human Lp(a). The precipitates obtained with sera from orang-utan and chimpanzee formed reactions of complete identity with the Lp(a) precipitate with human serum. When sera from Old World monkeys and human subjects were tested in wells next to each other, spurs developed between the 2 precipitates, indicating that Lp(a)-like lipoproteins in Old World monkeys have partial identity with human Lp(a). Lp(a) immunoreactive materials were identified in association with lipids by means of fat staining of the precipitates. On the other hand, reactants which could be precipitated with anti-human Lp(a) sera were not detectable in prosimians and New World monkeys. These results suggest that serum Lp(a)-like lipoprotein is phylogenetically acquired in Old World monkeys. However, the possibility that the structures of serum Lp(a)-like lipoproteins in prosimians and New World monkeys are too different to react with anti-human Lp(a) sera cannot be ruled out.

Transient changes of serum lipoprotein(a) as an acute phase protein.

Serum lipoprotein(a) (Lp(a)) was serially determined after acute attacks of myocardial infarction and after surgical operations. Acute phase proteins, such as C-reactive protein, alpha 1-acid glycoprotein, alpha 1-antitrypsin and haptoglobin, increased rapidly and markedly after the episodes. Initial values of serum Lp(a) concentrations were almost the same in both groups. Increases in serum Lp(a) levels were also observed during the first few days, with a return to the initial levels after more than 1 month. The periods for reaching maximal levels of acute phase proteins were similar in both groups of patients. On the contrary, the period required for Lp(a) to reach the maximal level in the myocardial infarction group was significantly longer than in the post-operative group. The present study suggests that Lp(a) has the characteristics of an acute phase reactant and may play an important role in recovery from tissue damage.

High and low density lipoprotein cholesterol in myocardial and cerebral infarction.

High density lipoprotein (HDL) and low density lipoprotein (LdL) cholesterol levels were measured in fasting blood samples from 950 healthy subjects and 188 aged patients by a new simple method. The HDL-cholesterol levels and HDL/LDL-cholesterol ratios are significantly higher in females than in males. In the healthy subjects, there are slight decreases in the levels of HDL-cholesterol and HDL/LDL-cholesterol ratio with aging in both sexes. The patients with myocardial infarction had significantly lower HDL-cholesterol levels and HDL/LDL-cholesterol ratios as compared to those of the group without infarction. On the contrary, no differences in total lipoprotein cholesterol levels were observed in the patients with cerebral infarction. The results, obtained in respect of electrocardiographic findings after the isoproterenol stress test, suggest that the HDL-cholesterol levels and HDL/LDL-cholesterol ratios may be related not only to the established myocardial infarction, but also to the presence of coronary atherosclerosis and stenosis.