Localization of Z-protein in isolated Z-disk sheets of chicken leg muscle

Immunoblotting studies with antisera against Z-protein, desmin, and alpha-actinin showed that Z-protein is clearly distinguishable from desmin and alpha-actinin. Z-protein is not a proteolytic product of another protein but is an intrinsic component of chicken breast muscle myofibrils. In these experiments, an SDS extract of intact muscle was first electrophoresed in a polyacrylamide gel, and then proteins were transferred to a nitrocellulose paper sheet. Detection of each protein on the sheet was made possible by the application of the indirect immunofluorescence technique with the respective antiserum. Immunofluorescence microscope studies using these antisera revealed that Z-protein has the same distribution as alpha-actinin in isolated Z- disk sheets. Anti-Z-protein antiserum and anti-alpha-actinin antiserum stained the interior of Z-disks. On the other hand, antiserum against desmin stained the periphery of Z-disks in isolated Z-disk sheets.     

A new Kupffer cell receptor mediating plasma clearance of carcinoembryonic antigen by the rat.

Previous studies were unable to identify Z-protein in elasmobranch liver with bromosulphophthalein as ligand. By using 8-anilinonaphthalene-1-sulphonate and Rose Bengal as ligands, however, we demonstrated in hepatic cytosol from Platyrhinoides triseriata an organic-anion-binding protein with gel-filtration characteristics identical with those of rat Z-protein. By comparison with pooled rat Z-protein, Pl. triseriata Z-protein had slightly lower affinity for 8-anilinonaphthalene-1-sulphonate and Rose Bengal, greatly decreased binding affinity for bromosulphophthalein and no binding activity for oleic acid or squalene. The Pl. triseriata Z-protein binding site was less hydrophobic than that of rat Z-protein. This observation may explain the differences in binding characteristics between the Z-proteins of these species.     

'O-Acyl isopeptide method' for the efficient preparation of amyloid beta peptide 1-42 mutants

Novel water-soluble isopeptides of Abeta1-42 mutants, '26-O-acyl isoAbeta1-42 (26-AIAbeta42) mutants', which were efficiently converted to intact Abeta1-42 mutants with no byproduct formation under physiological conditions, were synthesized. These isopeptides provide a new system useful for investigating the biological function of Abeta1-42 mutants.     

Endothelin receptors in human and guinea-pig gallbladder muscle

We measured contraction of muscle strips caused by endothelin (ET) isopeptides and binding of (125)I-ET-1 to muscle cell membranes prepared from human and guinea-pig gallbladders. Visualization of (125)I-ET-1 binding sites in tissue was performed by autoradiography. Results in human were similar to those in guinea-pig. ET-1 caused tetrodotoxin and atropine-insensitive contraction. The relative potencies for ET isopeptides to cause contraction were ET-1=ET-2>ET-3. ET-1 caused contraction was only slightly inhibited by BQ-123 (potent ET(A) receptor antagonist) and not by BQ-788 (potent ET(B) receptor antagonist). It was inhibited by the combination of both. Autoradiography localized (125)I-ET-1 binding to the smooth muscle layer. Binding of (125)I-ET-1 to muscle cell membranes was saturable and specific. Analysis of dose-inhibition curves demonstrated the presence of two classes of receptors. One class (ET(A) receptor) had a high affinity for ET-1 and ET-2 but a low affinity for ET-3, and the other (ET(B) receptor) a high affinity for ET-1, ET-2 and ET-3. These results demonstrate that similar to guinea-pig, human gallbladder possesses both ET(A) and ET(B) receptors cooperating to mediate muscle contraction.     

Cooling-induced contraction of guinea pig tracheal smooth muscle

Cooling of isolated guinea pig tracheal smooth muscle from 38 to 28 degrees C over 2.25 min produced a transient contraction followed by sustained relaxation. The cooling-induced contraction was blocked either by pretreatment with ouabain at concentrations of 10(-5) M or greater or by substitution of normal physiological salt solution with K-free solution. In contrast, the contractile response to cooling was not inhibited by pretreatment with phentolamine (10(-5) M), atropine (10(-5) M), tetrodotoxin (3 X 10(-7) M), diphenhydramine (10(-5) M), cromolyn sodium (10(-3) M), indomethacin (3 X 10(-7) M), nifedipine (10(-7) M), or verapamil (3 X 10(-6) M). Addition of NaHCO3 to the bath during cooling, preventing a change in pH of the physiological salt solution, did not affect the cooling-induced contraction. It is concluded that cooling of isolated guinea pig trachea produces a transient ouabain-sensitive contraction, and that the data suggest the contraction is mediated by inhibition of Na-K-ATPase in the smooth muscle rather than through neuronal stimulation or chemical mediator release.     

大鼠单一肌梭的电生理特征

本研究旨在观察大鼠单一肌梭的电生理特征.从大鼠比目鱼肌中分离单一肌梭,用空气隔绝法观察大鼠单一肌梭感觉末梢在不同液体环境中的放电活动.结果显示:在基础生理盐水溶液中,大鼠单一肌梭的自发放电频率很低,平均(51.78±25.63) impulse/1000s(n=13);在加有适量氨基酸的生理盐水溶液中,其自发放电频率明...     

Phosphorylation of the 20,000-dalton light chain of myosin of intact arterial smooth muscle in rest and in contraction.

The 20,000-dalton myosin light chain of intact pig carotid arteries was found to be rapidly labeled when the arterial muscle was incubated in physiological salt solution at 37 degrees C containing [32P]orthophosphate. Light chain phosphorylation in the intact muscle had a marked requirement for Ca2+ and was dependent upon the passive tension applied to the muscle. Norepinephrine- or KCl-induced contractures were associated with concomitant increases in light chain phosphorylation.     

Modulation of the inhibiting effect of long-chained acyl-CoA on oxidative phosphorylation of liver mitochondria in rats of various lines by Z-protein

The effect of short-term starvation on the energy state of the adenine nucleotide system in the livers of rats of Wistar and Wag strains was studied. In fed rats, (4 hours after food withdrawal) both strains had the same liver content of long-chain acyl-CoAs. At the same time, in the livers of Wistar rats the phosphate potential values were much higher than in the livers of Wag rats (1.82 +/- 0.18 and 0.64 +/- 0.08, respectively), thus suggesting a strong inhibition of oxidative phosphorylation in the livers of Wag rats. Based on a comparison of the states and levels of Z-protein in the livers of fed and starved Wistar and Wag rats, it was proposed that an excess of long-chain acyl-CoAs over the Z-protein content in the liver is a necessary prerequisite for the development of inhibition of oxidative phosphorylation. Thus, the Z-protein may be considered as a possible physiological modulator of the inhibitory effect of acyl-CoAs on the energy state of the adenine nucleotide system in the liver in vivo.     

Does endothelin mobilize calcium from intracellular store sites in rat aortic vascular smooth muscle cells in primary culture?

In the presence of endothelin, there was a rapid increase in the 45Ca++ efflux from primary cultured rat vascular smooth muscle cells, both in physiological salt solution and in calcium free medium containing 2 mM EGTA. The 45Ca++ influx was not affected. The endothelin-induced, transient increase in cytosolic calcium concentration is probably mainly due to release of calcium from the intracellular store in vascular smooth muscle cells.     

Cytosolic modulators of activities of microsomal enzymes of cholesterol biosynthesis. Effects of Acyl-CoA inhibition and cytosolic Z-protein

Physiological concentrations of long-chain fatty acyl-CoAs have now been shown to inhibit microsomal methyl sterol oxidase. Acyl-CoA inhibition of hydroxymethylglutaryl-CoA reductase as well as methyl sterol oxidase can be either prevented or reversed by the addition of purified Z-protein (fatty acid-binding protein). Concomitantly, Z-protein addition decreases the extent of binding of radioactively labeled oleoyl-CoA to microsomal membranes. Free heme also inhibits hydroxymethylglutaryl-CoA reductase, and Z-protein reverses the extent of observed inhibition by binding heme analogous to the effect observed with acyl-CoAs. Similarly, Z-protein reverses substrate inhibition of acyl-CoA:cholesterol acyltransferase at high concentrations of acyl-CoA substrate. All these observations are consistent with the suggestion that, by binding acyl-CoAs and other enzyme effectors such as free heme, Z-protein modulates the effects of fluctuations of concentrations of major cellular metabolites. Furthermore, because the concentration of Z-protein is very low in rapidly growing hepatomas, such tumors may be very poorly buffered against the effects of acyl-CoAs, free fatty acids, heme and other effectors that may vary markedly by either altered metabolism or release of metabolites from necrotic tumor tissue.     

Isolation of profilin from embryonic chicken skeletal muscle and evaluation of its interaction with different actin isoforms

An actin-binding protein of 16 kDa was isolated from embryonic chicken skeletal muscle. The protein had the same properties as profilin, exhibited a much higher affinity for cytoskeletal (beta- and gamma-) actins than for sarcomeric (alpha-) actin in the embryonic muscle, and inhibited the polymerization of beta- and gamma-actins more efficiently in a physiological salt solution. These results indicate that the assembly of cytoskeletal and sarcomeric actins is regulated differently by profilin in the developing skeletal muscle, and that the former may not be involved in myofibril assembly.     

A trypsin-sensitive, heat-labile, N-ethylmaleimide-sensitive factor in adipocyte post-microsomal supernatant which affects the assay of adipocyte glycerol phosphate acyltransferase activities.

Addition of adipocyte 100 000 g post-microsomal supernatant to assays of glycerol phosphate acyltransferase in isolated mitochondria or microsomal fractions decreased activity at lower concentrations of palmitoyl-CoA. At higher concentrations of palmitoyl-CoA, activation was observed on addition of post-microsomal supernatant. The effect of post-microsomal supernatant to decrease activity at lower [palmitoyl-CoA] was abolished by heating or by trypsin treatment, and was also abolished by addition of N-ethylmaleimide to assays or by pretreatment of post-microsomal supernatant with N-ethylmaleimide. The stimulatory effect seen at higher [palmitoyl-CoA] was not sensitive to heat or trypsin treatment. The effect of post-microsomal supernatant at lower [palmitoyl-CoA] cannot be attributed to palmitoyl-CoA hydrolase activity. It was found that brief treatment of adipocyte mitochondria with low concentrations of trypsin was an effective way to remove contaminating microsomal glycerol phosphate acyltransferase activity. Adipocyte post-microsomal supernatant was more effective than an equivalent quantity of liver post-microsomal supernatant protein in decreasing adipocyte microsomal glycerol phosphate acyltransferase activity. The effects of the supernatants from both tissues were decreased by flavaspidic acid. Semi-purified Z-protein fraction from rat liver did not mimic the effect of adipocyte post-microsomal supernatant to decrease glycerol phosphate acyltransferase at lower [palmitoyl-CoA]. Post-microsomal supernatants obtained from noradrenaline-treated adipocytes were less effective than those from control cells in decreasing glycerol phosphate acyltransferase activity in microsomal fractions at lower [palmitoyl-CoA]. It is suggested that adipocyte cytosol may contain an acyl-CoA-binding protein or proteins differing from Z-protein in some respects. The physiological significance of the findings is briefly discussed.     

Actions of lipoxygenase metabolites in isolated rat lungs

Leukotrienes constrict smooth muscle and could be important for the regulation of the pulmonary circulation. We examined the production and action of lipoxygenase metabolites in isolated lungs, where we controlled the perfusing fluid used. Arachidonate injected into isolated rat lungs perfused with cell- and protein-free physiological salt solution caused a transient pressor response. Following indomethacin, arachidonate caused a delayed slow pressure rise followed by edema. The lung effluent contracted the guinea pig ileum. High-pressure liquid chromatography (HPLC) analysis of the perfusate demonstrated the presence of leukotrienes (LTC4 and LTD4). Diethylcarbamazine, a leukotriene synthesis inhibitor, prevented the slow pressure rise and edema seen after indomethacin plus arachidonate. In lungs perfused with cell- and protein-free physiological salt solution, LTC4, but not LTD4, caused a transient pressure rise followed by a sustained pressure rise. The sustained rise was abolished by a leukotriene-receptor blocker (FPL 55712) but not by indomethacin. In blood-perfused lungs, LTC4 caused only the transient pressure rise that was not blocked by FPL 55712. In lungs perfused with physiological salt solution containing albumin, LTC4 had no effect. We concluded that 1) perfused nonsensitized rat lungs produced LTC4 and LTD4; 2) LTC4 may be a major pulmonary vasoconstrictor; and 3) albumin binding limits the pressor effect of LTC4.     

An actin-depolymerizing protein in embryonic chicken skeletal muscle: purification and characterization

In embryonic skeletal muscle, a large amount of non-polymerized actin exists in the cytoplasm (Shimizu and Obinata [1986] J. Biochem. 99, 751-759). A 19-kDa protein (called 19K protein) which binds to G-actin was purified by sequential chromatography on DNase I-agarose, hydroxylapatite, SP-Sephadex, and Sephadex G-75, from the sarcoplasmic fraction of embryonic chicken skeletal muscle. This protein decreased the extent of actin polymerization at a steady state and increased the monomeric actin in a concentration-dependent fashion; it also caused quick depolymerization of F-actin, as determined by spectrophotometry at 237 nm, viscometry, DNase I inhibition assay, and electron microscopy. The molar ratio of 19K protein and actin interacting with each other was estimated to be 1:1. From these results, 19K protein was regarded as being actin depolymerizing protein. The amount of 19K protein in muscle decreased during development. The inhibitory action of 19K protein was removed by myosin or heavy meromyosin, and actin filaments were formed on the surface of myosin filaments when myosin filaments were added to a mixture of actin and 19K protein in a physiological salt solution. We propose that actin assembly is dually controlled in the developing muscle by the inhibitor(s) and an accelerator (myosin); this mechanism may enable the ordered assembly of actin and myosin in the early phase of myofibrillogenesis.     

A close structural relationship of rat liver Z-protein to cellular retinoid binding proteins and peripheral nerve myelin P2 protein

Striking sequence homologies were found among the major cytosolic lipid and dye binding protein (Z-protein), cellular retinoid binding proteins and peripheral nerve myelin P2 protein. In addition, the presence of a number of cytosolic lipid binding (or exchange) proteins having similar molecular weights and amino acid compositions suggests a new family of intracellular proteins with high affinities to lipids, possibly diverged from a common ancestor. In view of regulatory roles of these proteins in lipid metabolism and transport, myelin P2 protein may also possess a similar physiological function(s).     

内皮素－1对血管平滑肌细胞增殖作用的研究进展（综述）

内皮素（ＥＴ）是迄今所发现的最强的内源性血管收缩肽,它有三种异构体,其中ＥＴ－１不仅缩血管活性最强,而且对血管平滑肌细胞（ＶＳＭＣ）具有丝裂原作用。本文从ＥＴ－１的合成和分泌,对ＶＳＭＣ的增殖作用以及丝裂原信息传递途径三方面,综述了目前ＥＴ－１对ＶＳＭＣ增殖作用的研究进展。     

Neuromedin U elicits cytokine release in murine Th2-type T cell clone D10.G4.1

Neuromedin U (NmU), originally isolated from porcine spinal cord and later from other species, is a novel peptide that potently contracts smooth muscle. NmU interacts with two G protein-coupled receptors designated as NmU-1R and NmU-2R. This study demonstrates a potential proinflammatory role for NmU. In a mouse Th2 cell line (D10.G4.1), a single class of high affinity saturable binding sites for (125)I-labeled NmU (K(D) 364 pM and B(max) 1114 fmol/mg protein) was identified, and mRNA encoding NmU-1R, but not NmU-2R, was present. Competition binding analysis revealed equipotent, high affinity binding of NmU isopeptides to membranes prepared from D10.G4.1 cells. Exposure of these cells to NmU isopeptides resulted in an increase in intracellular Ca(2+) concentration (EC(50) 4.8 nM for human NmU). In addition, NmU also significantly increased the synthesis and release of cytokines including IL-4, IL-5, IL-6, IL-10, and IL-13. Studies using pharmacological inhibitors indicated that maximal NmU-evoked cytokine release required functional phospholipase C, calcineurin, MEK, and PI3K pathways. These data suggest a role for NmU in inflammation by stimulating cytokine production by T cells.     

Z-protein, a component of the skeletal muscle Z-line, is located at the apical tips of microvilli of chicken intestinal epithelial cells

Antiserum against Z-protein, which is one of the components of myofibrillar Z-lines, strongly stained the epithelial cells of chicken small intestine at the apical tips of the microvilli and the junctional complexes. Immunoblot tests with anti-Z-protein antiserum showed that there is a peptide of the same antigenicity as myofibrillar Z-protein in the components of the epithelial cells of chicken small intestine. Hence it seems that the apical tip of the microvillus contains a constituent corresponding to the Z-protein of myofibrillar Z-line.     

THE COAGULATION OF MYOSIN IN MUSCLE

1. Muscle can be prepared in the form of a dry powder in which myosin exists in a state similar to that in intact muscle. As in intact muscle, myosin in powdered muscle is soluble and can be caused to rapidly coagulate. 2. Restoring to powdered muscle the quantity of water previously removed causes coagulation of myosin. The rate of coagulation is considerably slower at 0° than at 20°. 3. Adding the powder to a large volume of dilute salt solution also results in coagulation. 4. The water soluble constituents of muscle can be removed from the powder without thereby causing coagulation. Coagulation occurs in water extracted muscle when it is suspended in a dilute salt solution. 5. Coagulation of myosin in muscle resembles the coagulation of myosin caused by dehydration. 6. Myosin coagulates readily only when it is imbedded in the structure of muscle. The significance for coagulation of the arrangement of myosin particles in muscle has been indicated.     

Effects of palytoxin on contractile response and calcium movement in guinea-pig taenia coli

PTX (10(-8)M) induced a rapid increase followed by a gradual decrease in muscle tension in normal physiological salt solution (PSS), while it induced a slow increase in muscle tension in low-Na+ solution. These contractions were inhibited by Ca2+ channel blockers, verapamil and nicardipine. PTX rapidly increased tissue Na+ and decreased tissue K+ contents in normal PSS. In low-Na+ solution, PTX decreased tissue K+ content with a slower rate than that in normal PSS. PTX increased uptake of 45Ca2+ in normal as well as low-Na+ solutions with similar time course as the increase in muscle tension. However, 45Ca2+ uptake still remained high when the PTX-induced transient contraction ceased. These results suggest that PTX increases Ca2+ influx through voltage-dependent Ca2+ channels to cause contraction. After a prolonged exposure to PTX, however, muscle tension is uncoupled from Ca2+ influx.