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Biomedical subjects

T Toyofuku

Publications and source records attributed to T Toyofuku.

At least 37 records · Page 2Linked to original sources
PubMed1994

Amino acids Glu2 to Ile18 in the cytoplasmic domain of phospholamban are essential for functional association with the Ca(2+)-ATPase of sarcoplasmic reticulum.

Phospholamban inhibits the Ca(2+)-ATPase of cardiac sarcoplasmic reticulum by lowering its affinity for Ca2+. In earlier studies (Toyofuku, T., Kazimierz, K., Tada, M., and MacLennan, D. H. (1993) J. Biol. Chem. 268, 2809-2815), parts of the phosphorylation and nucleotide binding/hinge domains of the Ca(2+)-ATPase were shown to be essential for phospholamban interaction. In order to identify the sites in phospholamban which interact with the Ca(2+)-ATPase, a series of mutants containing amino acid replacements in the cytoplasmic and transmembrane regions of phospholamban were co-expressed with the cardiac/slow-twitch muscle Ca(2+)-ATPase isozyme, SERCA2a, in HEK-293 cells. Mutation of residues in the cytoplasmic 1A domain of phospholamban, including positively charged residues, Lys3, Arg9, Arg13, and Arg14, negatively charged residue, Glu2, hydrophobic residues, Val4, Leu7, Ala11, Ile12, Ala15, and Ile18, and phosphorylation site residues, Ser16 and Thr17, resulted in loss of the inhibitory effect of phospholamban on Ca2+ transport by the Ca(2+)-ATPase. By contrast, mutation of Met1, Gln5, Tyr6, Thr8, Ser10, Glu19, or Met20 or of residues in the cytoplasmic 1B domain (Pro21 to Asn30) and of Cys41 in the transmembrane domain (Leu31 to Leu52) did not diminish the inhibitory effects of phospholamban on Ca2+ transport. These results suggest that a region essential for functional association of phospholamban with the Ca(2+)-ATPase lies in the cytoplasmic 1A domain of phospholamban.

Amino Acid Sequence↗
PubMed1993

Mutation of glutamate 309 to glutamine alters one Ca(2+)-binding site in the Ca(2+)-ATPase of sarcoplasmic reticulum expressed in Sf9 cells.

Sf9 cells infected with a baculovirus vector containing SERCA1 cDNA expressed immunoreactive rabbit fast-twitch muscle Ca(2+)-ATPase at levels up to 3 mg/liter. The microsomal fraction isolated from infected Sf9 cells catalyzed Ca2+ transport at rates 6-fold above control values. To obtain direct evidence for the postulate (Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H., et al. (1989) Nature 339, 476-478) that Glu309 contributes to a Ca(2+)-binding site in the transmembrane sector of the Ca(2+)-ATPase, Ca2+ binding to wild type and mutant (Glu309 to Gln) Ca(2+)-ATPases was measured. The wild type Ca(2+)-ATPase, expressed in Sf9 cells and purified using a monoclonal antibody bound to Sepharose beads, bound approximately 1.6-1.7 mol Ca2+/mol of enzyme at 2 microM Ca2+ in a buffer favoring the E1 conformation of the enzyme and at 10 microM Ca2+ in a buffer favoring the E2 conformation. Under identical conditions, the mutant Ca(2+)-ATPase bound less than 0.1 mol of Ca2+/mol of enzyme in E1 buffer, but 0.8 mol Ca2+/mol in the E2 buffer. In spite of the ability of the Glu309 to Gln mutant enzyme to bind about 1 mol of Ca2+/mol of enzyme, E2P formation was not inhibited by up to 100 microM Ca2+, and E1P formation from ATP and Ca2+ was not observed with up to 100 microM Ca2+ in intact microsomal vesicles from Sf9 cells. Nevertheless, with detergent-solubilized and purified mutant Ca(2+)-ATPases, E2P formation was inhibited with a K0.5 of 2 microM Ca2+. These observations are consistent with the view that a single intact Ca(2+)-binding site is present in the mutant Ca(2+)-ATPase, which is accessible to Ca2+ only from the lumenal side and only in the E2 conformation. Transition from E2 to E1-Ca2+ may occur during or following Ca2+ binding, accounting for the relatively high Ca2+ affinity and inhibition by Ca2+ of phosphorylation from Pi.

Animals↗
PubMed1993

Identification of regions in the Ca(2+)-ATPase of sarcoplasmic reticulum that affect functional association with phospholamban.

When the SERCA 2 isoform of the Ca(2+)-ATPase of cardiac and slow-twitch muscle sarcoplasmic reticulum was coexpressed with phospholamban in COS-1 cells, a reduction in Ca2+ affinity (measured as Ca2+ dependence of Ca2+ transport) of 0.2-0.3 pCa units was observed. This inhibitory effect was reversed by phosphorylation of phospholamban with cAMP-dependent protein kinase A. SERCA 1 and SERCA 3, were also expressed in COS-1 cells, alone and together with phospholamban. SERCA 1 had high Ca2+ affinity which was reduced upon coexpression with phospholamban, but SERCA 3 had lower Ca2+ affinity, which was unaltered by coexpression with phospholamban. To identify which regions of the Ca2+ ATPase sequence determine its functional interaction with phospholamban, chimeric Ca(2+)-ATPases between SERCA 2 and SERCA 3 were constructed and coexpressed with phospholamban. Measurement of Ca2+ affinities for a series of chimeras showed that two separate regions of the cytoplasmic domain of SERCA 2 were required for manifestation of a functional interaction between phospholamban and the Ca(2+)-ATPase. The first is a region between amino acids 336 and 412 in the phosphorylation domain, which corresponds to a phospholamban interaction site identified earlier (James, P., Inui, M., Tada, M., Chiesi, M., and Carafoli, E. (1989) Nature 342, 90-92). The second region is the nucleotide binding/hinge domain (amino acids 467-762) which determines high Ca2+ affinity for SERCA type pumps (Toyofuku, T., Kurzydlowski, K., Lytton, J., and MacLennan, D. H. (1992) J. Biol. Chem. 267, 14490-14496).

Amino Acid Sequence↗
PubMed1993

Expression of phospholamban mRNA during early avian muscle morphogenesis is distinct from that of alpha-actin.

We have studied the expression of phospholamban during the early development of chick embryos by in situ hybridization and have compared it to that of alpha-cardiac and alpha-skeletal actin. In adult cross-striated muscles there is only one phospholamban gene and it is expressed exclusively in the heart and slow muscles. In the heart phospholamban transcripts were first detected at stage 14 in the region of presumptive ventricle and at stage 20 in the atrium. In the myotomal portion of the somites phospholamban mRNA was first detected at stage 20, which lagged behind the appearance of the alpha-actins. In the limb rudiments all three mRNAs were barely detectable through stage 24, but increased by stage 28+. However, quantitative analysis of signal intensity at stage 28+ indicated that less phospholamban mRNA is present in the limb bud than in the myotome since for phospholamban the ratio of the signal density in the myotome to that in the limb rudiments was about twice the value of the ratio determined for the alpha-actins. Northern blot analysis of embryonic day 11 chick fast pectoralis muscle showed that phospholamban mRNA was not detected in vivo while alpha-cardiac actin mRNA was. Moreover, no phospholamban mRNA was detected in primary cultures derived from pectoralis muscle of the same age. In concert with previous observations that phospholamban is not detectable at stage 30-32 in wing or thigh muscle, these results suggest that phospholamban mRNA is expressed independently of the alpha-actins in the limb buds during early myogenesis.

Actins↗
PubMed1992

The nucleotide binding/hinge domain plays a crucial role in determining isoform-specific Ca2+ dependence of organellar Ca(2+)-ATPases.

Several isoforms of organellar Ca(2+)-ATPases have been identified, each of which is expressed in a tissue-specific manner. In order to examine the functional properties of fast-twitch (SERCA 1a), cardiac/slow-twitch (SERCA 2a), and non-muscle (SERCA 3) isoforms of the Ca(2+)-ATPase, cDNAs of each type were expressed transiently in COS-1 cells. A study of the Ca2+ dependence of Ca2+ uptake showed that SERCA 1 and SERCA 2 have identical Ca2+ dependences (K0.5 = pCa 6.87 +/- 0.03 and pCa 6.87 +/- 0.02, respectively), but SERCA 3 has a lower Ca2+ dependence (K0.5 = pCa 6.32 +/- 0.03). A study of the ATP dependence of Ca2+ uptake showed that SERCA 1, 2, and 3 have almost identical ATP dependences. Average Hill coefficients derived from Ca2+ uptake curves ranged from 1.7 to 1.8 for the three isoforms. In order to identify which regions of the linear sequence determine this difference in Ca2+ dependence, chimeric Ca(2+)-ATPases between SERCA 2 and SERCA 3 were constructed. Chimeric Ca(2+)-ATPases containing the nucleotide binding/hinge domain of SERCA 2 had SERCA 2 type Ca2+ dependence, but both nucleotide binding/hinge and COOH-terminal transmembrane domains of SERCA 3 were required for SERCA 3 type Ca2+ dependence. Accordingly, structural interactions between the nucleotide binding/hinge and COOH-terminal transmembrane domains appear to determine isoform-specific Ca2+ dependences.

Animals↗
PubMed1992

Expression of alpha-cardiac and alpha-skeletal actin mRNAs in relation to innervation in regenerating and non-regenerating rat skeletal muscles.

The expression of alpha-cardiac and alpha-skeletal actin mRNA in regenerating muscle was examined. Changes in mRNA levels were analyzed in autografted extensor digitorum longus (EDL) muscles in rats using alpha-isoform specific synthetic oligonucleotides and beta-actin cDNA as probes. After autografting, the expression of alpha-cardiac actin mRNA was induced; concomitantly that of alpha-skeletal actin mRNA was reduced. The pattern of alpha-actin mRNA expression appeared to be similar to that seen in embryonic skeletal muscle. In order to evaluate the effects of innervation on alpha-actin mRNA expression in regenerating muscle, nerveless, standard, and nerve-intact autografted muscles were examined. More complete innervation facilitated the recovery of alpha-skeletal actin mRNA to control levels, but had little effect on the amount of alpha-cardiac actin mRNA. We found that regenerating muscle shows that embryonic pattern of alpha-actin mRNAs in the early stage and concluded that the recovery of alpha-skeletal actin mRNA expression to the adult pattern is influenced by innervation, while alpha-cardiac actin mRNA expression is nerve independent.

Actins↗
PubMed1992

Dibutyryl cyclic AMP attenuates lung responses induced by endotoxin in conscious sheep.

Dibutyryl cyclic AMP (DBcAMP) could inhibit the production of prostanoids and modulate the pulmonary vascular responses induced by endotoxin. Diffuse lung injury after endotoxemia in sheep is accompanied by the production of prostanoids and an increase in endothelial permeability. To determine whether exogenous DBcAMP could prevent the endotoxin responses, we measured pulmonary hemodynamics, gas exchange, and lung lymph responses to an intravenous infusion of Escherichia coli endotoxin (1.0 micrograms/kg over 30 min) in unanesthetized sheep in the presence and absence of DBcAMP (30 micrograms/kg/min) infused intravenously for 6 h beginning 1 h before endotoxin infusion or for 4.5 h after 30 min of treatment with endotoxin infusion. We also measured circulating leukocytes and lung lymph and plasma concentrations of thromboxane B2 (TXB2) and prostacyclin (6-keto-PGF1 alpha) metabolites by radioimmunoassay. DBcAMP infusion before endotoxin infusion decreased endotoxin-induced pulmonary hypertension and hypoxemia and markedly attenuated the increased lung lymph flow and lymph protein clearance. DBcAMP after endotoxin only attenuated the increased lung lymph flow and lymph protein clearance. DBcAMP treatment both before and after endotoxin infusion blocked endotoxin-induced increases in lung lymph and plasma TXB2 and 6-keto-PGF1 alpha. DBcAMP did not affect the number of circulating leukocytes. Although DBcAMP alone did not affect the pulmonary and systemic hemodynamics and lung lymph balance, the potential that DBcAMP directly modulates the pulmonary vascular responses to endotoxin as a vasodilator could be expected. We conclude that DBcAMP infusion attenuates lung dysfunction caused by endotoxemia, possibly by preventing prostanoid release and modulating the pulmonary vascular responses.

6-Ketoprostaglandin F1 alpha↗
PubMed1991

Characterization of cDNA and genomic sequences encoding a chicken phospholamban.

We report on the isolation and characterization of cDNA and genomic sequences encoding a chicken cardiac phospholamban. Three mRNAs, 0.6, 1.1, and 3.3 kilobase pairs in size, were detected in cardiac and slow-tonic muscle RNAs on Northern blots. We determined that these mRNAs differ in the length of their 3'-untranslated regions, perhaps because they utilize alternative polyadenylation signals. We studied the developmental and tissue-specific expression of phospholamban mRNA using nuclease mapping analysis. There appeared to be considerable homology between coding and 3'-untranslated regions of phospholamban mRNAs from cardiac and slow-tonic muscles during development, strongly suggesting that these mRNAs are encoded by the same gene. The phospholamban gene, which is present as a single copy in the chicken genome, is about 10 kilobase pairs long, with approximately 6.5 kilobase pairs representing the intron sequences. Primer extension and nuclease mapping analyses of the 5' end of the phospholamban mRNA showed that the three transcripts are initiated from the same initiation site. Analysis of the putative promoter region revealed "TATA box" and "CAAT box" sequences, putative muscle-specific elements, and a cyclic AMP-responsive element.

Adenosine Triphosphatases↗
PubMed1990

Actin and myosin isoforms in aneural and malformed chick hearts.

Although it is generally accepted that actin and myosin isoforms adapt to their functional requirements, the sequence of expression of these proteins in hearts developing abnormally is unknown. In the chick embryo it is possible to change various aspects of heart development without direct manipulation of the cardiovascular system, by removing various regions of the neural crest from early embryos. The neural crest provides both neural (sympathetic and parasympathetic) and ectomesenchymal components to the heart, and selective removal of various areas results in embryos with sympathetically aneural hearts, or persistent truncus arteriosus with or without parasympathetic denervation. Myosin isoform expression was studied in each of these types of hearts using an array of myosin antibodies specific for atrium, ventricle or the conduction system. Myosin expression in experimental hearts was found to follow the normal pattern of development using these antibodies. Actin expression was studied using cDNA probes for the 3' untranslated region of actin mRNA of the alpha-skeletal, alpha-cardiac and beta-actin isoforms. Using slot-blot hybridization analysis, the pattern of actin expression in atrium and ventricle was followed throughout the period of incubation in normal hearts. The pattern of actin expression was found to be abnormal in hearts which were sympathetically aneural and those which had persistent truncus arteriosus combined with parasympathetic denervation. ATPase activity was increased only in atria of hearts with persistent truncus arteriosus. It appears from these experiments that actin isoform expression is influenced in the chick heart by autonomic innervation.

Actins↗
PubMed1989

Tetrazolium artifactually indicates superoxide dismutase-induced salvage in reperfused rabbit heart.

We tested the ability of a single dose of superoxide dismutase to induce salvage of reperfused rabbit myocardium. Infarct size was measured by tetrazolium method following 3, 24, or 72 h of reperfusion. In addition, the 24 h reperfused hearts were examined to determine if the drug induced salvage in those hearts was reflected in the histology. A coronary arterial branch was occluded for 45 min and then allowed to reperfuse for 3, 24 or 72 h. At the end of the reperfusion period the hearts were removed, perfusion stained with triphenyl tetrazolium, and fixed in buffered formalin. The hearts were sectioned and infarct size was determined in all groups. In addition, the 24 h heart slices were prepared for histology with H&E staining. The results revealed that 5 mg/kg hSOD treatment was associated with smaller infarcts in the 3 and 24 h groups but that differences were no longer apparent in the 72 h group. The 24 h control hearts showed good correlation between infarct size by TTC and that by conventional histology. In the 24 h treatment hearts, however, infarcts by TTC averaged only about 1/2 the size of those by conventional histology. We conclude that a single dose of hSOD fails to offer a sustained reduction of infarct size. Furthermore, histology from the 24 h reperfused group revealed that hSOD did not delay the onset of necrosis but rather simply caused dead tissue to retain its ability to reduce the tetrazolium salts.

Animals↗
PubMed1989

Effects of polycations on pulmonary vascular permeability in conscious sheep.

The role of charged sites on the permeability characteristics of the pulmonary microvascular barrier were investigated using chronically instrumented unanesthetized sheep. In one series of experiments we studied the effects of the cationic amphiphile, dodecyl trimethylamine (DTA; 297 mol wt), and the anionic amphiphile, SDS (288 mol wt), on lung lymph flow rates (Ql), lung lymph to plasma protein ratios (L/P), pulmonary hemodynamics, and systemic hemodynamics. DTA significantly increased both Ql and L/P, whereas SDS had a more modest and transient effect on these variables. In a second series of experiments the polycations polybrene and poly-l-lysine were found to have very similar effects as those of DTA. In another series of experiments we tested the pretreatment inhibition potential of chlorpheniramine (an H1 receptor antagonist), dibutyryl-cyclic AMP (db-cAMP), and the calcium channel antagonists verapamil and nifedipine on polybrene-induced lung injury. We found that only verapamil and db-cAMP significantly attenuated the permeability effects of polybrene. We conclude that both cationic amphiphiles and polycations cause hemodynamic and permeability alterations in the pulmonary circulation of unanesthetized sheep. In addition, the permeability alterations induced by polybrene can be modulated by intracellular calcium and/or cAMP levels.

Animals↗
PubMed1988

Pulmonary vascular response to platelet-activating factor in conscious sheep.

Hemodynamic response to an intravenous infusion of platelet-activating factor (PAF; 1 microgram/kg) was studied in conscious sheep with lung lymph fistulas. PAF induced increases in pulmonary arterial pressure and decreases in left atrial and systemic arterial pressures and in cardiac output, together with transient increases in thromboxane (Tx) A2 (as TxB2) and prostacyclin (as 6-keto-PGF1 alpha) values in plasma and lung lymph. There were also transient decreases in circulating leukocytes and platelets. The second infusion of PAF induced a reduced response compared with the first one, but the response to PAF was afterward maintained. Pretreatment with OKY 046, a thromboxane synthase inhibitor, reduced the first pulmonary vasoconstriction in response to PAF to a degree equivalent to that after successive infusions of PAF in control sheep. Pulmonary response to PAF (except on first infusion) and systemic cardiovascular response did not change whether or not TxA2 was produced. We therefore concluded that PAF mediates pulmonary and systemic cardiovascular effects via mechanisms other than TxA2 during successive infusions of PAF and that PAF-induced TxA2 contributes only to the pulmonary response to PAF on first infusion of the latter.

6-Ketoprostaglandin F1 alpha↗
PubMed1988

Effects of coronary ischemia on lung fluid balance in conscious sheep.

It has been suggested that coronary ischemia increases extravascular lung water. To determine whether pulmonary microvascular permeability is increased by coronary ischemia, we measured pulmonary hemodynamics, lung lymph flow (QL), and lymph-to-plasma protein concentration ratio (L/P) in 12 sheep with chronic lung lymph fistulas. Studies were done in 3 groups: in group 1 (n = 7) a marginal branch of the left circumflex artery (Lcx) was occluded, in group 2 (n = 5) left atrial pressure (Pla) was mechanically raised by 10 mmHg, and in group 3 (n = 5) Lcx was occluded and Pla was raised by 10 mmHg. In group 1, coronary occlusion increased QL (4.6 +/- 0.4 to 8.3 +/- 2.6 ml/h) without changes in L/P. In group 2, elevated Pla increased QL (5.1 +/- 1.2 to 10.1 +/- 3.0 ml/h) with decreases in L/P (0.71 +/- 0.02 to 0.61 +/- 0.02). In group 3, coronary occlusion with elevated Pla caused a further increase in QL (5.0 +/- 1.5 to 16.9 +/- 4.6 ml/h) without significant decreases in L/P (0.71 +/- 0.01 to 0.65 +/- 0.06). Lung lymph concentrations of 6-keto-prostaglandin F1 alpha (a degradation product of prostacyclin) increased transiently after coronary occlusion. These results indicate that coronary occlusion can increase transcapillary protein transport in lungs of conscious sheep and simultaneously increase prostacyclin production in the lung.

6-Ketoprostaglandin F1 alpha↗
PubMed1987

The effect of dextran solution on pulmonary plasma-lymph barrier in awake sheep.

We investigated the effect of dextran solution on lung lymph flow in awake sheep with chronic lung lymph fistulas. Ten percent of dextran (molecular weight 40,000) solution or normal saline were infused at 1000 ml/h for 2 h through left atrial catheter. We measured pulmonary arterial pressure (Ppa), left atrial pressure (Pla), aortic pressure (Psa), cardiac output (CO), oncotic pressures of both plasma (IImv) and lung lymph (IIpmv), lung lymph flow rate (Qlym), and lymph-to-plasma ratio of total protein (L/P). Infusion of dextran solution caused significant increases in Ppa, Pla and CO and decrease in plasma-lung lymph oncotic pressure gradient (IImv-IIpmv), without changes in L/P. Infusion of normal saline caused significant increases in Ppa and Pla and no changes in IImv-IIpmv and CO, and slight decrease in L/P. The calculated filtration coefficients increased by 2.2 fold after dextran infusion and 1.7 fold after normal saline infusion. Moreover, an apparent increase in protein transport across microvessels as evidenced by the normal L/P despite increases in hydrostatic pressure occurred after dextran solution infusion. These results suggest that dextran solution may increase permeability of microvascular wall, as well as effective net pressure gradient across microvessels, both of which result in a large net fluid filtration from microvessels to perimicrovascular compartment.

Animals↗