Stimulation of phospholipase-D in Epstein Barr virus-transformed human B-cells.
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B-Lymphocytes↗
Biomedical subjects
K A Foster
Publications and source records attributed to K A Foster.
Effect of Rb+ on cromakalim-induced relaxation and ion fluxes in guinea pig trachea.
The effects of cromakalim, verapamil and salbutamol have been examined in guinea pig trachealis smooth muscle in both Krebs physiological salt solution and Krebs solution where K+ has been replaced by Rb+. Cromakalim-induced relaxation in the presence of Rb+ was reduced in extent and became transient, whilst the relaxation response to verapamil was enhanced and that to salbutamol unaffected. The transient relaxation occurring in Rb+ was blocked by quinidine and glibenclamide. The presence of extracellular Rb+ also prevented cromakalim-stimulated efflux of both 86Rb+ and 42/43K+. There was, however, no effect on cromakalim-stimulated 86Rb+ uptake. It is proposed that cromakalim is opening two populations of potassium channel in guinea pig tracheal smooth muscle, one of which is susceptible to blockade by Rb+ and one of which is not. The latter channel appears to play the dominant role in cromakalim-stimulated uptake, and is responsible for the transient relaxation response in the presence of rubidium, whilst the former is responsible for the maintained relaxation.
Albuterol↗
Altered responsiveness of hypertrophied rat hearts to alpha- and beta-adrenergic stimulation.
Inotropic responsiveness to alpha- and beta-adrenergic agents was examined in pressure-overload hypertrophied rat hearts. Pressure overload was induced in rats by abdominal aortic constriction. Three weeks post-constriction, hearts were isolated and perfused with buffer containing various concentrations of (1) calcium (2) isoproterenol (3) forskolin, or (4) phenylephrine. The change in rate of left ventricular pressure development (delta + dP/dt) with increasing perfusate calcium concentrations was comparable in hypertrophied hearts of aortic-constricted rats (AC) and hearts of sham-operated rats (SO). However, with isoproterenol or forskolin stimulation, inotropic responsiveness (delta + dP/dt) was 50% lower in hypertrophied hearts of AC. This was associated with significantly lower tissue cAMP levels. Beta-adrenoceptor number and affinity were unchanged in the hypertrophied myocardium. Maximum inotropic responsiveness to phenylephrine was also lower in hypertrophied hearts and was associated with reduced alpha-adrenoceptor numbers. The data suggest that altered inotropic responsiveness to alpha-adrenergic stimulation may, in part, be due to reduced cardiac alpha-adrenoceptor density. However, post-receptor mechanisms including alterations in cAMP metabolism may contribute to the reduced responsiveness to beta-adrenergic stimulation in hypertrophied hearts of AC.
Adrenergic alpha-Agonists↗
Synthesis and smooth muscle relaxant activity of a new series of potassium channel activators: 3-amido-1,1-dimethylindan-2-ols.
The synthesis of a novel series of smooth muscle relaxants which have been shown to act via the opening or activation of potassium channels is described. Compounds have been evaluated for their ability to inhibit spontaneous tone in guinea pig isolated trachealis and structure-activity relationships are discussed. One compound in particular, 1,1-dimethyl-5-nitro-3-(2-pyridon-1-yl)indan-2-ol, (16) was identified as a potent relaxant of airways smooth muscle in vitro with IC50 = 0.15 microM and was found to significantly inhibit histamine-induced dyspnoea in conscious guinea pigs when given orally 30-45 min prior to challenge.
Amides↗
Relaxant activity of 6-cyano-2,2-dimethyl-2H-1-benzopyran-4-carboxamides and -thiocarboxamides and their analogues in guinea pig trachealis.
Structural modifications of the potassium channel activator cromakalim (1) are described in which the amide moiety at C-4 has been replaced by carboxamide and thiocarboxamide functions. Analogues in which the hydroxyl group at C-3 has been oxidized or removed are also disclosed. Such analogues display an interesting profile of smooth muscle relaxant activity in the guinea pig isolated trachea, not all of which appears to result from the opening of potassium channels, but few compounds retain useful in vivo activity. However, one compound in particular, 6-cyano-2,2-dimethyl-N-methyl-2H-1-benzopyran-4-thiocarboxamide (13) was shown to be a potent potassium channel activator in vitro and to provide prolonged protection to guinea pigs from the respiratory effects of inhaled histamine.
Animals↗
Purification, properties, and phosphorylation by protein kinase C of two phosphoinositidase C isozymes from rat brain.
Two forms of phosphoinositidase C have been purified from the soluble fraction of rat brain. The purification scheme included gel filtration followed by chromatography on cellulose phosphate, phenyl-Sepharose, and Mono Q. Gradient sodium dodecyl sulphate-polyacrylamide gel electrophoresis gave apparent molecular masses of 151 kDa and 147 kDa. Western blotting with monoclonal antibodies showed that the isozymes corresponded to PLC-beta-1 and PLC-gamma of bovine brain. With both enzymes phosphatidylinositol 4,5-bisphosphate was a better substrate than phosphatidylinositol at neutral pH and low calcium ion concentrations. Both enzymes produced a proportion of inositol 1:2-cyclic phosphates from each substrate, particularly at acid pH. Some GTPase activity was seen in the early stages of purification, but was separated from PLC-beta-1 and PLC-gamma on Mono Q. Purified rat brain protein kinase C phosphorylated PLC-gamma but not PLC-beta-1. Incubation with the kinase increased the activity of both enzymes however, possibly by phosphorylation of another protein in the preparations.
Animals↗
Effect of calcium and cAMP on Go alpha expression in neonatal rat cardiac myocytes.
Culturing neonatal rat cardiac myocytes in 50 mM KCl inhibits the accumulation of Go alpha that occurs when myocytes are placed in culture. The mechanism by which high extracellular K+ inhibits Go alpha accumulation in myocytes was investigated by measurement of the concentration of intracellular Ca2+ ([Ca2+]) and adenosine 3',5'-cyclic monophosphate concentration ([cAMP]) of control and K(+)-depolarized myocytes. Although intracellular [Ca2+] in K(+)-depolarized myocytes was twofold higher than basal intracellular [Ca2+] in control cells, the mean intracellular [Ca2+] in contracting control myocytes was comparable to that of K(+)-depolarized myocytes. Furthermore, myocytes cultured in low Ca2+ plus high K+ exhibited an inhibition of Go alpha accumulation, even though intracellular [Ca2+] was 10-fold lower than that of cells cultured in normal Ca2+ plus high K+. In addition, intracellular [cAMP] of K(+)-depolarized myocytes was comparable to that of control cells. Moreover, dibutyryl cAMP inhibited Go alpha accumulation in myocytes to the same extent as high K+, even though intracellular [cAMP] differed 10-fold. Thus neither intracellular Ca2+ nor cAMP appear to mediate the inhibitory effect of high K+ on Go alpha accumulation. However, cAMP has an inhibitory effect on Go alpha expression that is independent of K+.
Animals↗
Expression of G proteins in rat cardiac myocytes: effect of KCl depolarization.
As a first step in understanding the functioning of hormonal signaling pathways in the heart, the G protein composition of neonatal and adult rat hearts was determined by immunoblotting analysis. Neonatal rat cardiac myocytes and nonmuscle heart cells contained Gs alpha 52, Gi alpha 1,3, and Gi alpha 2 with little or no Gs alpha 45 or Go alpha 39. Interestingly, Go alpha 39 accumulated fourfold in cardiac myocytes between days 1 and 6 of culture. Atrial membranes from adult rat hearts had approximately equal amounts of the 45- and 52-kDa forms of Gs alpha, whereas adult ventricles had predominantly Gs alpha 45. In addition, adult atria contained relatively more Gi alpha 1,3, Gi alpha 2, and Go alpha 39 than adult ventricle. Moreover, the increase in Go alpha 39 observed in cardiac myocytes cultured for 6 days could be prevented by culturing cells in medium containing 50 mM KCl. The observed differences in G protein expression between cell types and between contracting and KCl-depolarized cardiac myocytes may provide a system to investigate the function of various G proteins and to study regulation of their expression.
Animals↗
The cloned polC gene of Bacillus subtilis: characterization of the azp12 mutation and controlled in vitro synthesis of active DNA polymerase III.
Wild type (wt) Bacillus subtilis polC and polCazp12, a mutant derivative specifying a form of DNA polymerase III resistant to hydroxyphenylazopyrimidines, were cloned as genomic fragments approximating the length required to encode the entire polymerase. The cloned DNA fragments were subjected to restriction and partial sequence analysis to locate the 5' end of the polC-specific coding sequence and the azp12 mutation, which was identified as a T----G transversion specifying replacement of serine with alanine. The cloned wt and azp12-coding sequences were recloned in an Escherichia coli expression vector with their respective 5' ends under the control of the bacteriophage lambda PL promoter and cIts857-encoded repressor. In response to induction, the wt- and azp12-specific recombinant plasmids expressed active DNA polymerases indistinguishable from the native enzymes derived from the respective B. subtilis hosts.
Amino Acid Sequence↗
Role of G proteins in the regulation of the cardiovascular system.
A precise description of the involvement of G proteins in regulation of the cardiovascular system is not possible at the present time although it is clear that they do have important regulatory roles. The cardiovascular system is composed of a variety of cell types, which are subject to control by several different hormones, as well as by hormones that have several different effects in the same cell type. Although, historically, variations in the type and number of receptors located on each cell have been used to explain this diversity of hormonal responses, we must now consider the large number and diversity of G proteins in any effort to understand the coordinated hormonal regulation of cellular functions. Given that there are eight known G proteins and several others have been speculated, each of which is composed of three subunits, each of which has several different forms, the possible combinations of subunits into functionally distinct G proteins is enormous. To place this newly described family of G proteins into the appropriate hormone signaling pathways will require a continued research effort. However, with recent progress in producing specific antibodies to each of the G protein subunits, it may now be possible to determine the specific receptor-effector functions of each G protein and their individual subunits.
Animals↗
Calcium-dependent calmodulin-binding proteins associated with mammalian DNA polymerase alpha.
Complex, multiprotein forms of bovine (calf thymus), hamster (Chinese hamster ovary cell), and human (HeLa) cell DNA polymerase alpha (Pol alpha) were analyzed for their content of calmodulin-binding proteins. The approach used an established autoradiographic technique employing 125I-labeled calmodulin to probe proteins in denaturing SDS-polyacrylamide gel electropherograms. All three Pol alpha enzymes were associated with discrete, Ca2+-dependent calmodulin-binding proteins. Conventionally purified calf thymus Pol alpha holoenzyme contained three prominent, trifluoperazine-sensitive species with apparent molecular masses of approx. 120, 80 and 48 kDa. The 120 and 48 kDa species remained associated with the polymerase.primase core of the calf enzyme during immunopurification with monoclonal antibodies directed specifically against the polymerase subunit. The patterns of the calmodulin-binding proteins displayed by conventionally purified preparations of hamster and human Pol alpha enzymes were similar to each other and distinctly different from the pattern of comparable preparations of calf thymus Pol alpha. Immunopurified preparations of the human and hamster Pol alphas retained significant calmodulin-binding activity of apparent molecular masses of approx. 55, 80 and 150-200 kDa.
Animals↗
Inhibition of mammalian 5-lipoxygenase by 2-benzylaminophenols.
A number of 2-benzylaminophenols, prepared from the corresponding 2-aminophenols by reductive alkylation, have been identified as highly potent inhibitors of 5-lipoxygenase with IC50 values in the nanomolar range. Most compounds were also shown to inhibit the release of the peptidoleukotrienes when administered intraperitoneally in a rat model of peritoneal anaphylaxis. Two compounds evaluated for their effects on anaphylactic contractions in isolated human lung were shown to attenuate the leukotriene-induced component of the response.
Aminophenols↗
Novel 1H-benzimidazol-4-ols with potent 5-lipoxygenase inhibitory activity.
The synthesis and structure--activity profile of 2-substituted benzimidazol-4-ols as inhibitors of cell-free RBL-1 5-lipoxygenase are discussed, and their potency is compared with that of the standard inhibitors phenidone, AA 861, BW 755C, and nordihydroguaiaretic acid. In contrast to the standard compounds, most did not inhibit the release of slow-reacting substance of anaphylaxis (SRS-A) in vivo when administered at 200 microM ip to rats subjected to peritoneal anaphylaxis, although five compounds containing a methoxylated benzyl group (compounds 36, 39, 42, and 43) or hydroxylated benzyl group (41) showed similar activity to that of phenidone, nordihydroguaiaretic acid, and AA 861. Of the many compounds tested, two, 5-tert-butyl-7-methyl-2-(trifluoromethyl)-1H-benzimidazol-4-ol (57) and 2-(4-methoxybenzyl)-7-methyl-1H-benzimidazol-4-ol (36), like dexamethasone, inhibited monocyte accumulation in a pleural exudate model of inflammation. Standard lipoxygenase inhibitors such as phenidone, BW 755C, and AA 861 were inactive in this system.
Animals↗
Mechanisms for altered carnitine content in hypertrophied rat hearts.
Carnitine levels are reduced in hypertrophied hearts of rats subjected to aortic constriction (banding) and elevated in hypertrophied hearts of spontaneously hypertensive rats (SHR). In an attempt to determine the mechanisms for these alterations, L-[14C]carnitine transport was examined in isolated perfused hearts. Total carnitine uptake was significantly reduced by approximately 20% in hypertrophied hearts of banded rats at all perfusate carnitine concentrations employed. The reduction in total uptake was due to a 40% reduction in carrier-mediated carnitine uptake with no difference in uptake by diffusion. In contrast, carnitine uptake was not altered in isolated hypertrophied hearts of SHR. However, serum carnitine levels were elevated in SHR, which could result in increased myocardial carnitine uptake in vivo. The data suggest that altered carnitine content in hypertrophied hearts of aortic-banded rats is due to an alteration in the carrier-mediated carnitine transport system in the myocardium. However, altered carnitine content in hypertrophied hearts of SHR is not due to a change in the carnitine transport system per se but may rather be due to a change in serum carnitine levels.
Animals↗
Altered phospholipid metabolism in pressure-overload hypertrophied hearts.
The content and fatty acyl composition of phospholipids were examined in pressure-overload hypertrophied hearts. Cardiac hypertrophy was induced in rats by abdominal aortic constriction. Twenty-one days postconstriction the content of myocardial phosphatidylcholine (PC), sphingomyelin, and phosphatidylinositol (PI) was significantly elevated by 10, 10, and 20%, respectively. The essential fatty acid, linoleic acid, was markedly reduced in PC, phosphatidylethanolamine (PE), PI, and cardiolipin (CL) of hypertrophied hearts. The associated changes in fatty acyl composition were specific for the individual phospholipid class as evidenced by a significant elevation of palmitic acid in PC, docosahexaenoic acid in PE and oleic acid in CL. Alterations in fatty acyl composition of phospholipids were associated with no change in the composition of cardiac triglycerides, cardiac free fatty acids or serum lipids. The fatty acyl composition of phospholipids was also altered in pressure-overload hypertrophied hearts of cats, as evidenced by a reduction of linoleic acid and an elevation of arachidonic acid in total phospholipids. These findings demonstrate that changes in phospholipid metabolism occur in the pressure-overloaded mammalian heart. Such alterations may contribute to altered membrane function in the hypertrophied myocardium.
Animals↗
The interrelation between DNA synthesis rates and DNA polymerases bound to the nuclear matrix in synchronized HeLa cells.
Quantitative rates of DNA synthesis can be determined by DNA:propidium fluorescence measurements of synchronized cells progressing through S-phase. We have previously reported that HeLa cells have discontinuous rates with values of about 2.9, 1.6, and 4.4 pg of DNA/h for early, middle, and late S-phase, respectively. In attempts to understand why two peaks of DNA synthesis rates are observed, we have examined the nuclear DNA polymerases alpha and beta over the S-phase. Nuclear matrices isolated from HeLa cells contained 2% of the alpha polymerase and 12% of the beta polymerase that was present in cell lysates, and about 2% of the original DNA. The amounts of endogenous DNA synthesis in isolated nuclear matrices were comparable to the amounts observed when exogenous DNA was added. DNase treatment abolished the endogenous DNA synthesis but not the exogenous DNA synthesis, suggesting that polymerase alpha binding does not depend on matrix-bound DNA. As synchronized cells progressed through the S-phase, there appeared two peaks of enzymatic activity of alpha polymerase bound to the nuclear matrix which correlated with in vitro DNA synthesis in these nuclear matrices and with the two peaks of quantitative DNA synthesis rates. Two peaks of alpha polymerase activity were also observed with isolated nuclei, but not with cell lysates or cytosol. Our results suggest that, over the S-phase, the differential binding of polymerase alpha to the nuclear matrix determines the differential rates of DNA synthesis.
Cell Nucleus↗
Altered carnitine metabolism in spontaneously hypertensive rats.
Carnitine metabolism was examined in spontaneously hypertensive rats (SHR). Carnitine levels were elevated by 25% in hypertrophied hearts of 10- and 15-wk-old SHR when compared with Wistar-Kyoto (WKy) controls. This elevation was associated with a greater than 25% increase in total serum carnitine. The elevated serum carnitine does not appear to be due to increased mobilization from skeletal muscle because carnitine levels were elevated by 25% in gastrocnemius and diaphragm of SHR. Elevated serum carnitine is also not a result of reduced urinary excretion because daily urinary carnitine output was increased by 150% in SHR. These findings suggest that the most likely mechanism for increased serum carnitine is increased carnitine synthesis by the liver. The changes in carnitine metabolism in SHR appear to occur between 5 and 10 wk of age, because the carnitine levels in serum and organs were comparable in 5-wk-old WKy and SHR. The observed alterations in tissue and serum carnitine levels may result in altered fatty acid utilization in SHR.
Animals↗
Coronary vascular responsiveness to non-eicosanoid vasoconstrictors in the perfused diabetic rat heart.
We tested arginine vasopressin, and a dihydropyridine calcium agonist, BAY K-8644 in isolated perfused hearts from control and diabetic rats. Arginine vasopressin (1-100 ng/ml) and BAY K-8644 (100-500 ng/ml) significantly increased coronary perfusion pressure during constant flow perfusion indicative of coronary vasoconstriction. However, no significant potentiation was observed between diabetic rats and their weight matched controls for either vasoconstrictor.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗