bluuebunny/crossref_metadata_2025 huggingface dataset:
Investigation of neutral endopeptidases (EC 3.4.24.11) and of neutral proteinases (EC 3.4.24.4) using a new sensitive two‐stage enzymatic reaction
Authors: Indig Fred E. , Ben-Meir Daniella , Spungin Anya , Blumberg Shmaryahu
Published: July 2002

A sensitive two‐stage enzymatic reaction for mammalian and bacterial metalloendopeptidases has been developed using the substrate 3‐carboxypropanoyl‐alanyl‐alanyl‐leucine‐4‐nitroanilide supplemented with Streptomyces griseus amino‐peptidase. Neutral endopeptidase EC 3.4.24.11 from bovine kidney hydrolyzes the substrate (pH 7.5, 25°C) with a catalytic efficiency (k
cat=1.2 × 102 s−1, K
m=0. 15 mM) of the highest ever reported for the enzyme acting on synthetic chromophoric and fluorogenic substrates. Thermolysin hydrolyzes the substrate at a faster rate (k
cat=1.2 × 103 s−1) but the overall efficiency is diminished by a higher K
m (4.2 mM). Suspensions of human neutrophil cells and culture filtrates of Bacillus cereus have been assayed sensitively for their neutral endopeptidase and neutral proteinase activities, respectively. The assay provides a convenient tool for the kinetic investigation of neutral endopeptidases and neutral proteinases and for assessing their function in biological systems.
Mitochondrial porin regulates the sensitivity of anion carriers to inhibitors
Authors: Pronevich Ludmila A. , Mirzabekov Tajib A. , Rozhdestvenskaya Zinaida E.
Published: July 2002

In mitoplasts, respiratory stimulation by ADP, palmitate, DNP and CCCP and sensitivity of respiration to carboxyatractylate are considerably less pronounced than in mitochondria. Addition of porin‐containing preparations (purified outer membranes or solubilized mitochondrial porin) to mitoplasts results in partial restoration of the oxygen consumption and sensitivity to carboxyatractylate (CAT). The uncoupling effect of FCCP in mitoplasts is CAT‐resistant and does not depend on added porin. It is suggested that mitochondrial porin may be a natural activator of ADP/ATP antiporter and succinate carrier in mitochondria.
Tyrphostins inhibit the epidermal growth factor receptor‐mediated breakdown of phosphoinositides
Authors: Posner Israel , Gazit Aviv , Gilon Chaim , Levitzki Alexander
Published: July 2002

In response to epidermal growth factor (EGF) and the Ca2+ ionophore A23187, the total phosphatidylinositides (IPT) increased in A431 human epidermoid carcinoma cells 1.8‐ and 2.0‐fold and in the EGF‐dependent A431/Clone 15‐2 cells 3.0‐ and 8.0‐fold, respectively, over basal levels. Both responses were inhibited by the antiproliferative agents tyrphostins, but the EGF‐induced increase in IPT was inhibited to a much greater extent than that induced by the ionophore. Tyrphostins which are potent EGF‐receptor kinase inhibitors were also potent in blocking the EGF‐induced production of phosphoinositides. The less potent tyrphostins were found to inhibit the EGF‐dependent IPT formation more weakly. These results support the notion that phospholipase C is activated through its phosphorylation by the EGF receptor. Tyrphostins; Epidermal growth factor; Phospholipase C phosphorylation; Ca2+ ionophore; (A431 cell, A431/Clone 15‐2 cell)
Wheat germ agglutinin but not concanavalin A modulates protein kinase C‐mediated phosphorylation of red cell skeletal proteins
Authors: Danilov Yuri N. , Cohen Carl M.
Published: July 2002

Human red blood cells contain protein kinase C (PKC) which acts exclusively on the membrane skeletal proteins band 4.1, band 4.9 and adducin. PKC activity can be stimulated by the addition of the phorbol ester 12‐O‐tetradecanoyl phorbol 13‐acetate to intact cells. Phosphorylation of band 4.1 by PKC in vitro results in a dramatic reduction in band 4.1 binding to spectrin and actin, as well as to the cytoplasmic domain of band 3. Here we show that the lectin wheat germ agglutinin (WGA), which binds to the extracellular domain of glycophorin results in the inhibition of PKC catalyzed phosphorylation of band 4.1, band 4.9 and likely adducin as well. The lectin concanavalin A, which binds to band 3 was without effect. Our results suggest that the binding of WGA to glycophorin results in a major rearrangement of the membrane skeletal network which correlates with reduced phosphorylation of membrane skeletal proteins by PKC.
Advances in Na+,K+‐ATPase studies: from protein to gene and back to protein
Authors: Broude N.E. , Modyanov N.N. , Monastyrskaya G.S. , Sverdlov E.D.
Published: July 2002

Complete primary structures of both subunits of Na+,K+,ATPase from various sources have been established by a combination of the methods for molecular cloning and protein chemistry. The gene family homologous to the α‐subunit cDNA of animal Na+,K+‐ATPases has been found in the human genome. Some genes of this family encode the known isoforms (αI and αII) of the Na+,K+‐ATPase catalytic subunit. The proteins coded by other genes can be either new isoforms of the Na+,K+‐ATPase catalytic subunit or other ion‐transporting ATPases. Expression of the genes of this family proceeds in a tissue‐specific manner and changes during the postnatal development and neoplastic transformation. The complete exon‐intron structure of one of the genes of this family has been established. This gene codes for the form of the catalytic subunit, the existence of which has been unknown. Apparently, all the genes of the discovered family have a similar intron‐exon structure. There is certain correlation between the gene structure and the proposed domain arrangement of the α‐subunit. The results obtained have become the basis for the experiments which prove the existence of the earlier unknown αIII isoform of the Na+,K+‐ATPase catalytic subunit and have made possible the study of its function.
Immunoaffinity isolation of Na,K‐ATPase α3 isoform from pig kidney
Authors: Arystarkhova E.A. , Lakhtina O.E. , Levina N.B. , Modyanov N.N.
Published: July 2002

ATPase isozyme, (Na+ + K+)‐; Immunoaffinity chromatography; N‐terminal sequence analysis
Inhibition of epidermal growth factor‐induced DNA synthesis by tyrosine kinase inhibitors
Authors: Umezawa Kazuo , Hori Takashi , Tajima Hirohisa , Imoto Masaya , Isshiki Kunio , Takeuchi Tomio
Published: July 2002

We prepared methyl 2,5‐dihydroxycinnamate as a stable analogue of erbstatin, a tyrosine kinase inhibitor. This analogue was about 4 times more stable than erbstatin in calf serum. It inhibited epidermal growth factor receptor‐associated tyrosine kinase in vitro with an IC50 of 0.15 . It also inhibited in situ autophosphorylation of epidermal growth factor receptor in A431 cells. Methyl 2,5‐dihydroxycinnamate was shown to delay the S‐phase induction by epidermal growth factor in quiescent normal rat kidney cells, without affecting the total amount ofDNA synthesis. The effect of erbstatin on S‐phase induction was smaller, possibly because of its shorter life time.
On the role of insulin in regulation of adenosine deaminase activity in rat tissues
Authors: Rutkiewicz Joanna , Górski Jan
Published: July 2002

After administration of insulin adenosine deaminase activity was reduced in different skeletal muscle types, the heart and the liver. On the other hand profound reduction in the plasma insulin concentration (streptozotocin diabetes) resulted in elevation of the enzyme activity in the tissues. It is concluded that the local concentration of adenosine may be effected by the concentration of insulin in the plasma.
Characterization of Na+‐independent Mg2+ efflux from erythrocytes
Authors: Günther T. , Vormann J.
Published: July 2002

Na+‐independent Mg2+ efflux from Mg2+‐loaded human, rat and chicken erythrocytes was reduced by extracellular Cl−. Na+‐independent Mg2+ efflux at low extracellular Cl− concentration (sucrose medium) was inhibited by SITS and was nearly insensitive to SITS in 150 mM choline Cl medium. The inhibition of Mg2+ efflux by extracellular Cl− and DIDS could be overcome by the lipophilic permeant tetraphenylphosphonium cation. Na+‐independent Mg2+ efflux from human and rat erythrocytes in sucrose and choline Cl medium was inhibited by cAMP and by amiloride and amiloride analogues. The results indicate that Na+‐independent Mg2+ efflux in high Cl− medium is performed by a similar or the same Mg2+ efflux system, operating in sucrose medium in which the efflux of Mg2+ is accompanied by the efflux of Cl− for charge compensation.
Ca2+‐mobilising properties of synthetic fluoro‐analogues of myo‐inositol 1,4,5‐trisphosphate and their interaction with myo‐inositol 1,4,5‐trisphosphate 3‐kinase and 5‐phosphatase
Authors: Safrany Stephen T. , Sawyer Deborah , Wojcikiewicz Richard J.H. , Nahorski Stefan R. , Potter Barry V.L.
Published: July 2002

The ability of two fluoro‐analogues of D‐myo‐inositol 1,4,5‐trisphosphate (Ins(1,4,5)P3) to mobilize intracellular Ca2+ stores in SH‐SY5Y neuroblastoma cells has been investigated. DL‐2‐deoxy‐2‐fluoro‐scyllo‐Ins(1,4,5)P3 (2F‐Ins(1,4,5)P3) and DL‐2,2‐difluoro‐2‐deoxy‐myo‐Ins(1,4,5)P3 (2,2‐F2‐Ins(1,4,5)P3) were full agonists (EC50s 0.77 and 0.41 μM respectively) and slightly less potent than D‐Ins(1,4,5)P3 (EC50 0.13 μM), indicating that the axial 2‐hydroxyl group of Ins(1,4,5)P3 is relatively unimportant in receptor binding and stimulation of Ca2+ release. Both analogues mobilized Ca2+ with broadly similar kinetics and were substrates for Ins(1,4,5)P3 3‐kinase but, qualitatively, were slightly poorer than Ins(1,4,5)P3. 2F‐Ins(1,4,5)P3 was a weak substrate for Ins(1,4,5)P3 5‐phosphatase but 2,2‐F2‐Ins(1,4,5)P3 was apparently not hydrolysed by this enzyme, although it inhibited its activity potently (Ki = 26 μM).
Potentiation of angiotensin II‐stimulated phosphoinositide hydrolysis, calcium mobilization and contraction of renal mesangial cells upon down‐regulation of protein kinase C
Authors: Pfeilschifter Josef , Fandrey Joachim , Ochsner Martin , Whitebread Steven , de Gasparo Marc
Published: July 2002

Long‐term pretreatment of rat mesangial cells with 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) down‐regulated protein kinase C activity and potentiated the angiotensin II‐induced inositol trisphosphate (InsP3) formation. This increased response to angiotensin II occurred without a significant change in the receptor number or K
d value of angiotensin II binding to the cells. The biologically inactive phorbol ester 4α‐phorbol 12,13‐di‐decanoate was without effect on angiotensin II‐stimulated InsP3 generation. Long‐term pretreatment with TPA also increased the angiotensin II‐induced mobilization of Ca2+ and the subsequent contraction of mesangial cells.
Palytoxin acidifies chick cardiac cells and activates the Na+/H+ antiporter
Authors: Frelin Christian , Vigne Paul , Breittmayer Jean-Philippe
Published: July 2002

The cardiotoxic action of palytoxin was investigated using embryonic chick ventricular cells. Under normal ionic conditions, palytoxin produced an intracellular acidification which is partially compensated for by the Na+/H+ antiporter thereby leading to an increased rate of ethylisopropylami‐loride‐sensitive 22Na+ uptake. Under depolarizing membrane conditions, palytoxin produced a cellular acidification, a cellular alkalinization or no change in intracellular pH depending on the value of the extracellular pH. We propose that palytoxin acidifies cardiac cells by opening preexisting H+ conducting pathways in the plasma membrane.
Direct characterization of the Na+/H+ exchanger in human platelets
Authors: Astarie C. , David-Dufilho M. , Devynck M.A.
Published: July 2002

The kinetic properties of the Na+/H+ exchanger in human platelets were investigated by direct measurements of pHi as detected with the fluorescent dye, BCECF. In acid‐loaded cells, the antiporter displayed a hyperbolic dependence regarding external Na+ with an apparent K
m of 38 ± 4 mM (pHo 7.2 at 25°C) whereas its pHi‐dependent activation between 7.3 to 6.4 did not obey a Michaelian model. External acidification from 7.7 to 6.5 decreased significantly the initial rate of Na+‐dependent H+ efflux. The amiloride derivative, ethylisopropylamiloride blocked this exchanger and exerted a non‐competitive inhibition with respect to Na+
o (K
i = 17 nM). The cation selectivity of the external site of the antiporter was Na+ > Li+ > K+ and choline. These results indicate that the BCECF technique allows to evaluate the main features of the Na+/H+ exchanger in human platelets, which possesses kinetic properties similar to those reported in other cell types.
3,4‐Dichloroisocoumarin, a serine protease inhibitor, inactivates glycogen phosphorylase b
Authors: Rusbridge Nicholas M. , Beynon Robert J.
Published: July 2002

3,4‐Dichloroisocoumarin (3,4‐DCI) is a highly reactive, mechanism‐based inhibitor of serine proteases. We show here that glycogen phosphorylase b is also inactivated by this inhibitor, in a mechanism that parallels the inactivation of serine proteases, but involving multiple sites of covalent modification. Such a process may compromise studies in which 3,4‐DCI is used to arrest proteolysis of a second native protein which may itself be modified.
Pi trapping in glycogenolytic pathway can explain transient Pi disappearance during recovery from muscular exercise
Authors: Bendahan David , Confort-Gouny Sylviane , Kozak-Reiss Geneviève , Cozzone Patrick J.
Published: July 2002

31P NMR spectroscopy at 4.7 T has been used to follow changes in phosphorylated metabolites and pHi in the flexor digitorum superficialis muscle of 15 healthy volunteers subjected to a rest‐exercise‐recovery protocol. Phosphomonoesters (Pme) increased during exercise and exhibited a delayed recovery to resting level. During early recovery, Pi fell below resting concentration without correlated PCr oversynthesis while Pme level stayed above its resting value. The sum Pi + Pme remained constant. These observations suggest that Pi could be trapped into the glycogenolytic pathway during exercise leading to Pme production. This trapping and the slow Pme recovery could account for transient Pi disappearance observed during recovery.
Okadaic acid uncouples myosin light chain phosphorylation and tension in smooth muscle
Authors: Tansey M.G. , Hori M. , Karaki H. , Kamm K.E. , Stull J.T.
Published: July 2002

Tracheal smooth muscle precontracted with carbachol relaxes upon the addition of 3 μM okadaic add. Although cytosolic Ca2+ concentrations decrease, myosin light chain remains highly phosphorylated (50%). In smooth muscle treated with carbachol alone or carbachol plus okadaic acid 32P is incorporated into a single peptide on myosin light chain which corresponds to the site phosphorylated by myosin light chain kinase. Treatment with okadaic acid alone does not result in myosin light chain phosphorylation or tension development. These results suggest that a cellular mechanism other than myosin light chain phosphorylation can regulate contractile tension.
Protein kinase C in insulin releasing cells
Authors: Wollheim Claes B. , Regazzi Romano
Published: July 2002

The evidence for the involvement of protein kinase C (PKC) in insulin secretion stimulated by glucose and Ca2+‐mobilizing receptor agonists has been reviewed. Results of phorbol ester binding to intact cells and the measurements of the proportion of PKC associated with the membrane after cell fractionation are presented. Glucose stimulation leads to increased phorbol ester binding without causing membrane insertion of the enzyme which, however, occurs with receptor agonists. It is suggested that the rise in cytosolic Ca2+ in response to glucose favours the apposition of PKC to the membrane whereas intercalation of the enzyme requires phospholipase C‐mediated generation of diacylglycerol. It is possible that this effect of glucose on PKC, although not involved in the initiation of secretion, could explain the potentiation of insulin release observed in the presence of the receptor agonists.
Simultaneous recording of Indo‐1 flourescence and Na+/Ca2+ exchange current reveals two components of Ca2+‐release from sarcoplasmic reticulum of cardiac atrial myocytes
Authors: Lipp Peter , Pott Lutz , Callewaert Geert , Carmeliet Edward
Published: July 2002

Simultaneous measurements of intracellular Ca2+‐concentration ([Ca2+]1) using Indo‐1 and the current generated by electrogenic Na1/Ca2+‐exchange (INa???
have been performed on atrial myocytes from hearts of adult guinea‐pigs. Whereas the flourescence‐measurements provide information on global[Ca2+]i, INa???
which is a linear function of Ca2+‐concentration, indicates subsarcolemmal [Ca2+]. Under conditions in which intracellular Ca2+‐transients due to Ca2+‐release from the sarcoplasmic reticulum (SR) have been artificially slowed, a deviation between the two different Ca2+‐signals can be found. During onset of release signals Ca2+‐concentration seen by the membrane is higher than global [Ca2+]1. Our results provide evidence that in atrial myocytes, lacking a T‐system, Ca2+‐release occurs first from a subsarcolemmal compartment of the SR. The resulting Ca2+‐transient serves to trigger Ca2+‐release from deeper SR‐compartments.
The role of carbohydrates in vectorial exocytosis The secretion of the gp 80 glycoprotein complex in a ricin‐resistant mutant of MDCK cells
Authors: Parczyk Karsten , Koch-Brandt Claudia
Published: July 2002

In the polarized epithelial Madin‐Darby canine kidney (MDCK) cell line an 80 kDa glycoprotein complex (gp 80) is sorted into the spical pathway or exocytosis and is secreted constitutively at the apical cell surface. The unglycosylated form or the protein complex is secreted in a nonpolar fashion at both surface domains [(1987) J. Cell. Biol. 105, 2735–2743]. Using ricin‐resistant MDCK cells the role of the terminal galactose and slalic acid residues in the sorting of the gp 80 complex was analysed. The results suggest that the carbohydrate cores, rather than the ultimate or penultimate sugar residues, play a critical role in the, intracellular transport of this protein.
Swimming training increases the G4 acetylcholinesterase content of both fast ankle extensors and flexors
Authors: Gisiger Victor , Sheker Shauna , Gardiner' Phillip F.
Published: July 2002

The effect of endurance swimming training on AChE molecular forms was examined in 2 groups of functionally antagonist rat muscles, including ankle extensors and flexors, This exercise regimen, which entails predominant dynamic activity (i.e., involving extensive shortening) of both groups or muscles, resulted in marked selective G4 increases in all fast muscles. The G4 elevation exhibited by the ankle flexors was in sharp contrast to the G4 reduction reported in these same muscles following running training, during which their action is predominantly tonic. The results strengthen the conclusion that predominantly dynamic activity increases the G4 content of mature innervated fast muscles.
D‐[35S(U)]Inositol 1,4,5‐trisphosphorothioate, a novel radioligand for the inositol 1,4,5‐trisphosphate receptor Complex binding to rat cerebellar membranes
Authors: Challiss R.A.John , Smith Susan M. , Potter Barry V.L. , Nahorski Stefan R.
Published: July 2002

D‐[35S(U)]mya‐inositol 1,4,5‐trisphosphorothioate ([35S]InsPS3), a synthetic, metabolically stable analogue of inositol 1,4,5‐trisphosphate (InsP3) binds with high affinity (Ka
58.6±9.1 nM) to rat cerebellar revealing a high density of specific binding sites (D
max 21.5±2.1 pmol/mg of protein). Comparison with [3H]InsP3 binding reveals a higher density of sites labelled by [35S]InsPS3 and complex competition curves for displacement of specific [35S]insPS3 by InsP3. The results suggests that [35S]InsPS3 labels two sites in rat cerebellar membranes with equal affinity: the InsP3 receptor and a site that displays low affinity for InsP3.
cAMP‐dependent protein kinase activation affects vasopressin V2‐receptor number and internalization in LLC‐PK1 renal epithelial cells
Authors: Jans David A. , Hemmings Brian A.
Published: July 2002

The relationship between activation of the cAMP‐dependent protein kinase (cAMP‐PK) and ligand binding and internalization by the vasopressin renal (V2‐type) receptor of LLC‐PK1 renal epithelial cells was examined. Upon cAMP‐PK activation through 1 h treatment with the cAMP analogue 8‐bromo‐cAMP (BrcA), a marked reduction in V2‐receptor steady state number and internalization in LLC‐PK1 cells was effected. In cells treated for 17 h with BrcA and hence down‐regulated for cAMP‐PK1 the V2‐receptor number was normal but internalization was markedly reduced. Cells of the LLC‐PK1 mutant FIB4, which possesses about 10% parental cAMP‐PK catalytic subunit activity, exhibited lower V2‐receptor steady state number and internalization in comparison to untreated LLC‐PK1 cells. A negative correlation was thus evident between cAMP‐PK activation and V2‐receptor number and internalization. Phosphorylation by cAMP‐PK may effect ligand‐independent removal of receptor from the plasma membrane.
Is the mitochondrial Ca2+ uniporter a voltage‐modulated transport pathway?
Authors: Kapùs András , Szászi Katalin , Káldi Krisztina , Ligeti Erzsébet , Fonyó Attila
Published: July 2002

The influence of membrane potential (Δψ) on Ca2+ transport through the Ca2+ uniporter (UP) was investigated in fura‐2‐loaded rat heart mitochondria at physiologically relevant‐submicromolar‐external [Ca2+]. In the absence of Δψ the UP could not mediate Ca2+ uptake even when an 8‐fold external (∼500 nM) to internal (∼60 nM) [Ca2+] gradient was present and charge compensation was provided by acetate and the protonophore, CCCP. A small (∼−120 mV) and transient Δψ (generated by valinomycin) resulted in a rise in matrix [Ca2+] only when external [Ca2+] exceeded 150 nM. At physiologically high (∼−180 mV) and stable Δψ this threshold value for Ca2+ uptake dropped to 15 nM. The results indicate that (1) at physiological [Ca2+]m Δψ in addition to being a component of ΔμCa2+
seems to be necessary for providing a transport‐competent conformation for the UP; and (2) below a threshold [Ca2+]m the UP cannot operate even in the presence of a high electric driving force.
Altered glucose 1,6‐bisphosphate and fructose 2,6‐biphosphate levels in low‐frequency stimulated rabbit fast‐twitch muscle
Authors: Green Howard J. , Cadefau Juan , Pette Dirk
Published: July 2002

Glucose 1,6‐bisphosphate (Glc‐1,6‐P2) and fructose 2,6‐bisphospate (Fru‐2,6‐P2) concentration display pronounced increases in rabbit fast‐twitch muscle during chronic low‐frequency stimulation. These increases are first seen after stimulation periods exceeding 3 h and reach maxima after 12–24h of stimulation (approximately 3‐fold for Glc‐1,6‐P2 and 5‐fold for Fru‐2,6‐P2). Both matabolites regress to normal values after stimulation periods longer than 4 days. The fact that their increases coincide with the replenishment of glycogen after its initial depletion, could point to a role of Glc‐1,6‐P2 and Fru‐2,6‐P2 in glycogen metabolism.
Alpha 2‐macroglobulin synthesis in interleukin‐6‐stimulated human neuronal (SH‐SY5Y neuroblastoma) cells Potential significance for the processing of Alzheimer β‐amyloid precursor protein
Authors: Ganter Ursula , Strauss Sylvia , Jonas Uwe , Weidemann Andreas , Beyreuther Konrad , Volk Benedikt , Berger Mathias , Bauer Joachim
Published: July 2002

Cultured human neuronal (SH‐SY5Y neuroblastoma) cells synthesize and secrete the potent protease inhibitor alpha 2‐macroglobulin (a2M) upon stimulation with interleukin‐6(IL‐6) indicating that alpha 2‐macroglobulin behaves as an acute‐phase protein in the human central nervous system. Exogenous addition of a2M to the cultured neuronal cells resulted in only a slight inhibition of Alzheimer βA4‐amyloid precursor protein (APP) synthesis, but markedly inhibited its secretion pointing to the possibility that a2M may affect the proteolytic APP processing. Evidence is provided that IL‐6 and a2M are involved in Alzheimer's disease pathogenesis.
Cell volume is a major determinant of proteolysis control in liver
Authors: Häussinger Dieter , Hallbrucker Christian , vom Dahl Stephan , Decker Sheila , Schweizer Ursula , Lang Florian , Gerok Wolfgang
Published: July 2002

Hepatic proteolysis is inhibited by insulin, amino acids and hypoosmotic cell swelling and is stimulated by glucagon. These effectors simultaneously modulate cell volume in the intact liver, as shown by measurements of the intracellular water space. A close relationship exists between the effect on proteolysis and the accompanying cell volume change, regardless of whether hepatic proteolysis was modified by insulin, glucagon, cyclic AMP, glutamine, glycine, barium of hypoosmotic exposure. It is suggested that cell volume changes exerted by hormones and amino acids play a crucial role in the regulation of hepatic proteolysis.
Native Xenopus oocytes express two types of muscarinic receptors
Authors: Davidson Ariane , Mengod Guadalupe , Matus-Leibovitch Noa , Oron Yoram
Published: July 2002

We have recently described two types of muscarinic responses in native Xenopus oocytes of different donors (common and variant) that display qualitative and quantitative differences (Lupu‐Meiri et al., 1990). Here we characterized the muscarinic receptors mediating these two types. The anti‐muscarinic toxins from Dentroaspis significantly inhibited responses in oocytes of common donors, but had little effect on responses in oocytes of variant donors, possibly indicating expression of different receptor subtypes. Using specific muscarinic antagonists, we found that oocytes of common donors exhibit a pattern compatible with the M3 subtype of muscarinic receptors, while oocytes of variant donors appear to possess receptors of the M1 subtype. To more directly determine the subtypes of muscarinic receptors in oocytes of both populations of donors, we have microinjected antisense oligonucleotides into native oocytes. Antisense oligonucleotides to unique sequences in the N‐terminal and the third cytoplasmic loop of M3 muscarinic receptors caused a significant inhibition of the response of common oocytes, but had virtually no effect on responses in oocytes of variant donors. Conversely, oligonucleotides complementary to the unique sequences of the m1 muscarinic receptors inhibited the response in variant oocytes, but not in oocytes of common donors. We conclude that native Xenopus oocytes of different donors phenotypically express either M3‐like (majority) or M1‐like (minority) muscarinic receptor subtypes. The differences in receptor subtype expression may explain the different characteristics of responses in the two populations.
Vasoconstrictor‐induced protein‐tyrosine phosphorylation in cultured vascular smooth muscle cells
Authors: Tsuda Terutaka , Kawahara Yasuhiro , Shii Kozui , Koide Masanobu , Ishida Yoshihiro , Yokoyama Mitsuhiro
Published: July 2002

In cultured rat aortic smooth muscle cells, angiotensin II induced tyrosine phosphorylation of at least 9 proteins with molecular masses of 190, 117, 105, 82, 79, 77, 73, 45 and 40 kDa in time‐ and dose‐dependent manners. Other vasoconstrictors such as [Arg]vasopressin, 5‐hydroxytryptamine and norepinephrine induced the tyrosine phosphorylation of the same set of proteins as angiotensin II. The tyrosine phosphorylation of these proteins was mimicked by the protein kinase C‐activating phorbol ester, phorbol 12 myristate, 13‐acetate, and the Ca2+ ionophore, ionomycin. These results demonstrate that the vasoconstrictors stimulate the tyrosine phosphorylation of several proteins in vascular smooth muscle cells and suggest that the tyrosine phosphorylation reactions are the events distal to the activation of protein kinase C and Ca2+ mobilization in the intracellular signalling pathways of the vasoconstrictors.
Identification of the site phosphorylated by casein kinase II in smooth muscle caldesmon
Authors: Wawrzynow Alicja , Collins John H. , Bogatcheva Natalia V. , Vorotnikov Alexander V. , Gusev Nikolai B.
Published: July 2002

Phosphorylation of avian gizzard caldesmon by casein kinase II was investigated. The enzyme incorporates about 1 mol of phosphate per mol of caldesmon. All sites of phosphorylation are located in short chymotryptic peptides with M
r, 25–27 kDa or in the short N‐terminal peptide formed after cleavage of chicken gizzard caldesmon at Cys153. The primary structure of the tryptic peptide containing the main site of duck gizzard caldesmon phosphorylation is S‐E‐V‐N‐A‐Q‐N‐X‐V‐A‐E‐D‐E‐T‐K. where X is an unidentified residue. presumed to be phosphorserine. Thus, Ser73 is the main site phosphorylated by casein kinase II in avian gizzard caldesmon.
A cyclic‐AMP‐gated conductance in cochlear hair cells
Authors: Kolesnikov Stanislav S. , Rebrik Tatyana I. , Zhainazarov Aslbek B. , Tavartkiladze George A. , Kalamkarov Grigorii R.
Published: July 2002

The patch clamp technique was used to record cAMP‐dependent currents of the guinea pig cochlear hair cell plasma membrane. Data obtained indicate that the channels passing this current are moderately selective for monovalent cations and are effectively blocked by L‐cis‐diltiazem and reversibly blocked by 1 mM Mg2+ or Ca2+. The single‐channel unit conductance estimated in the absence of divalent cations is about 16 pS. The results demonstrate that cyclic nucleotide‐dependent channels of cochlear hair cells are virtually identical to the photoreceptor and olfactory ones.
Striking structural and functional similarities suggest that intestinal sucrase‐isomaltase, human lysosomal α‐glucosidase and Schwanniomyces occidentalis glucoamylase are derived from a common ancestral gene
Authors: Naim Hassan Y. , Niermann Thomas , Kleinhans Ulrich , Hollenberg Cornelis P. , Strasser Alexander W.M.
Published: July 2002

Sequence comparison of the primary structure of the yeast Schwanniomyces occidentalis glucoamylase (GAM) with GAMs in different microorganisms did not reveal significant similarities. By contrast, striking similarities were, surprisingly, found with 3 mammalian secretory and integral membrane proteins: the 2 subunits of intestinal brush border sucrase‐isomaltase and human lysosomal α‐glucosidase. The similarities among these proteins are found as clusters of up to 8 amino acids and distributed all over the protein sequences. The major sequence differences are found in the N‐terminal regions accounting, probably, for the different cellular locations of these proteins. The high level of similarities between sucrase, isomaltase, Sch. occidentalis GAM and human lysosomal α‐glucosidase suggest that these proteins are derived from the same ancestral gene. To our knowledge, this is the first report that describes similarities between a yeast secretory protein and mammalian secretory and integral membrane proteins.
Characterization and differential expression of protein kinase C isoforms in PC12 cells Differentiation parallels an increase in PKC beta11
Authors: Wooten Marie W. , Seibenhener M.Lamar , Soh Yunjo , Ewald Sandra J. , White Kimberly R. , Lloyd Elizabeth D. , Olivier Andree , Parker Peter J.
Published: July 2002

Nerve growth factor (NGF) treatment of PC12 cells induced a 2.8‐fold increase in protein kinase C activity concomitant with differentiation and acquisition or neurites. PKC protein isoforms were separated by sequential chromatography on DEAE‐Sephacel/hydroxylapatite. A broad peak or PKC activity eluted which corresponded to the alpha PKC isoform. In control cells, message for all six PKC isoforms was detected and expressed as epsilon>zeta=gamma>delta>beta>alpha. Western blot or whole cell lysates revealed a large increase in the beta11, while slight changes were observed for the other five PKC isoforms during treatment (12‐14 days) with NGF (50 ng/ml). In parallel, coordinate changes in the expression of the individual transcripts for the six isoforms occurred during NGF treatment. Induction and accumulation of PKC beta11 may play a role in maintenance or neuronal morphology.
Thromboxane receptor stimulation inhibits adenylate cyclase and reduces cyclic AMP‐mediated inhibition of ADP‐evoked responses in fura‐2‐loaded human platelets
Authors: Sage Stewart O. , Heemskerk Johan W.M.
Published: July 2002

Stimulation of human platelets with the thromboxane A2 analogue, U46619, after treatment with prostaglandin E1 or forskolin, reduced the inhibition of ADP‐evoked Mn2+ influx and the release of Ca2+ from intracellular stores, U46619 decreased the elevated concentration of 3′, 5′‐cyclic AMP in platelets that were pretreated with prostaglandin E1. These results suggest that occupation of prostaglandin H2/thromboxane A2 receptors. like those for other agonists, inhibits adenylate cyclase activity, which can contribute to the promotion of platelet activation.
Functional evidence of a transmembrane channel within the Ca2+ transport ATPase of sarcoplasmic reticulum
Authors: de Meis Leopoldo , Inesi Giuseppe
Published: July 2002

Ca2+ efflux can be studied conveniently following dilution of sarcoplasmic reticulum (SR) vesicles preloaded with 45Ca2+ by active transport. The rates of efflux are highly dependent on ATPase substrates and cofactors (Pi, Mg2+, Ca2+ and ADP) in the efflux medium. On the other hand, pbenothiazines stimulate efflux through a passive permeability channel with no coupled catalytic events. Efflux activation by manipulation of catalytically active ATPase ligands, as well as by the catalytically inactive phenothiazines, can be prevented by thapsigargin, which is a highly specific inhibitor of the Ca2+‐ATPase. This demonstrates that the passive channel activated by phenothiazines is an integral part of the ATPase, and can operate either uncoupled or coupled to catalytic events.
Cloning and characterization of the rat glutathione peroxidase gene
Authors: Ho Ye-Shih , Howard Adriann J.
Published: July 2002

The increased activity of glutathione peroxidase (GSHPx) in rat lungs is associated with the development of tolerance of the animals to hyperoxia. To understand further the regulation of expression of this enzyme, the molecular structure of the corresponding rat gene was characterized. The rat GSHPx gene consists of two exons interrupted by a single intron of 217 base pairs. The same initiation sites for transcription were found to be utilized in both lung and liver. The promoter of the GSHPx gene contains neither a ‘TATA’ box nor a ‘CAAT’ box. Instead, it comprises two copies of Sp1 binding motif and one copy of AP‐2 binding motif. These features of the promoter may offer a clue to the mechanisms by which the expression of this gene is controlled.
Structural and functional correlates of a mutation in the malignant hyperthermia‐susceptible pig ryanodine receptor
Authors: Mickelson Janies R. , Knudson C.Michael , Kennedy Catharine F.H. , Yang Ding-I. , Litterer Lynn A. , Rempel William E. , Campbell Kevin P. , Louis Charles F.
Published: July 2002

The skeletal muscle ryanodine receptor of malignant hyperthermia‐susceptible (MHS) pigs contains a mutation at residue 615 that is highly correlated with various abnormalities in the regulation of sarcoplasmic reticulum (SR) Ca2+ channel activity. In isolated SR membranes the Arg615 to Cys615 ryanodine receptor mutation is now shown to be directly responsible for an altered tryptic peptide map, due to the elimination of the Arg615 cleavage site. Furthermore, trypsin treatment released 86–99 kDa ryanodine receptor fragments encompassing residue 615 from the SR membranes. We conclude that the 86–99 kDa domain containing residue 615 is near the cytoplasmic surface of the ryanodine receptor and likely near important Ca2+ channel regulatory sites.
Nitrendipine is a potent inhibitor of the Ca2+‐activated K+ channel of human erythrocytes
Authors: Ellory J.Clive , Kirk Kiaran , Culliford Steven J. , Nash Gerard B. , Stuart John
Published: July 2002

Nitrendipine, a classical blocker of L‐type Ca2+ channels, is shown to be a potent inhibitor of the Ca2+‐activated K+ channel of human erythrocytes. In erythrocytes suspended in a solution with physiological Na+ and K+ concentrations and in which the channel was activated using the Ca2+ ionophore ionomycin, nitrendipene inhibited K+(80Rb+) influx with an I
50 of around 130 nM. Similar results were obtained for K+(80Rb+) efflux, and for K+(80Rb+) influx into cells suspended in a high‐K+ medium.
Native‐type DHP‐sensitive calcium channel currents are produced by cloned rat aortic smooth muscle and cardiac α1 subunits expressed in Xenopus laevis ooeytes and are regulated by α2‐ and β‐subunits
Authors: Itagaki Kiyoshi , Koch Walter J. , Bodi Ilona , Klöckner Udo , Slish Donald F. , Schwartz Arnold
Published: July 2002

Native tissue‐like L‐type voltage‐dependent calcium channels (L‐VDCC's) were expressed by in vitro transcribed cRNA injection of rat aorta or rabbit cardiac α1 subunit into Xenopus laevis oocytes. Co‐injection of VSM‐α1 with the cloned skeletal muscle β‐subunit (SK‐β) of the L‐type VDCC significantly increased the expressed peak current amplitude without significant changes in kinetics. Similar results were obtained by co‐injection of cardiac α1 (DSHT‐α1) the cloned skeletal α2‐subunit (SK‐α2) or with SK‐β. The oocytes co‐expressing cRNA's retained L‐type VDCC pharmacology.
Extracellular 2‐chloroadenosine and ATP stimulate volume‐sensitive Cl− current and calcium mobilization in human tracheal 9HTEo− cells
Authors: Galietta Luis J.V. , Rasola Andrea , Rugolo Michela , Zottini Michela , Mastrocola Teresa , Gruenert Dieter C. , Romeo Giovanni
Published: July 2002

The perforated‐patch whole‐cell technique was used to record membrane currents in epithelial cells (9HTEo−) obtained from the human tracheal epithelium. Extracellular application of 2‐chloroadenosine and ATP (0.01–100 μM) caused activation of Cl− currents similar to those regulated by cell volume in airway and intestinal cells. This response was inhibited by increasing extracellular osmolality, by omission of extracellular Ca2+ or by the addition of the A2 adenosine receptor antagonist 3,7‐dimethyl‐1‐propargylxanthine (DMPX). Fluorimetric measurements with fura‐2 reveal that 2‐chloroadenosine and ATP elicited both a Ca2+ influx through the plasma membrane and a release from intracellular stores.
Volume‐activated Cl− secretion and transepithelial vinblastine secretion mediated by P‐glycoprotein are not correlated in cultured human T84 intestinal epithelial layers
Authors: McEwan G.T.A. , Hunter J. , Hirst B.H. , Simmons N.L.
Published: July 2002

The relationship between the P‐glycoprotein‐mediated vinblastine secretion and cell‐swelling activated Cl− secretion (conductance) in intact epithelial layers of human colonic adenocarcinoma T84 cells has been investigated. Whereas vinblastine secretion is effectively inhibited by 100 μM 1,9‐dideoxy‐forskolin, volume‐stimulated Cl− secretion is unaffected. In contrast, 100 μM 4,4′‐diisothiocyanostilbene‐2,2′disulphonic acid (DIDS) inhibited the volume‐stimulated Cl− secretion, but was without effect upon transepithelial vinblastine secretion. In addition, it was noted that some epithelial layers failed to express a volume‐stimulated Cl− secretion but maintained a normal level of secretory vinblastine flux.
The differential role of Cys‐421 and Cys‐429 of the Glut1 glucose transporter in transport inhibition by p‐chloromercuribenzenesulfonic acid (pCMBS) or cytochalasin B (CB)
Authors: Wellner M. , Monden I. , Keller K.
Published: July 2002

Cys‐421 and Cys‐429 of Glut1 were replaced by site‐directed mutagenesis in order to investigate their involvement in basal glucose transport and transport inhibition. Neither of the two cysteine residues was essential for basal 2‐deoxy‐D‐glucose uptake in Xenopus oocytes expressing the respective mutant M421 and M429. If applied from the external side, the poorly permeable sulfhydryl‐reactive agent pCMBS inhibited 2‐deoxy‐D‐glucose uptake of Glut1 ‐ and M421‐expressing Xenopus oocytes but failed to affect uptake of the Cys‐429 mutant. This is in agreement with the proposed two‐dimensional model or Glut1 confirming that Cys‐429 is the only residue exposed to the surface of the plasma membrane. The replacement of Cys‐421 at the exofacial end of helix eleven caused a partial protection of 3‐O‐methylglucose transport inhibition by CB; this residue may thus be involved in stabilizing an adjacent local tertiary structure necessary for the full activity of this inhibitor.
Cyclic AMP‐dependent phosphorylation and regulation of the cardiac dihydropyridine‐sensitive Ca channel
Authors: Yoshida Akira , Takahashi Masami , Nishimura Seiichiro , Takeshima Hiroshi , Kokubun Shinichiro
Published: July 2002

A polyclonal antibody, CR2, prepared using the C‐terminal peptide of the α1 subunit of the rabbit cardiac DHP‐sensitive Ca channel, specifically immunoprecipitated the [3H]PN200‐100‐labeled Ca channel solubilized from cardiac microsomes. The antibody recognized 250 and 200‐kDa cardiac microsomal proteins as determined by immunoblotting, and cAMP‐dependent protein kinase phosphorylated the 250‐kDa, but not the 200‐kDa protein in vitro. CHO cells, transfected with the cardiac subunit cDNA carried by an expression vector, synthesized a 250‐kDa protein which was recognized by CR2. Adding db‐cAMP or forskolin to the transformed CHO cells induced phosphorylation of the 250‐kDa proteina and stimulated the DHP‐sensitive Ba current under patch‐clamp conditions. These results suggested that the cardiac DHP‐sensitive Ca channel was regulated by cAMP‐dependent phosphorylation of the α1 subunit.
Kinetic characterization of the acid—alkaline transition in Dolabella auricularia ferric myoglobin using 1H‐NMR saturation transfer experiments
Authors: Yamamoto Yasuhiko , Chûjô Riichirô , Inoue Yoshio , Suzuki Tomohiko
Published: July 2002

The acid—alkaline transition in ferric myoglobin of the mollusc, Dolabella auricularia, exerts the changes in both the coordination and spin states of the heme iron. Slower transition rate, compared to the NMR time scale, in this myoglobin allowed the observation of separate signals arising from the two forms, and pH tiltration yielded a pK value of 7.8. 1H‐NMR saturation transfer experiments have been successfully used not only to provide the first signal assignments for the heme methyl proton resonances of the Met‐hydroxyl form of the myoglobin, but also to detemine the kinetics of the transition.
Pulsed magnetic field effects on calcium signaling in lymphocytes: Dependence on cell status and field intensity
Authors: Walleczek Jan , Budinger Thomas F.
Published: July 2002

The effect of 3‐Hz, monopolar, quasi‐rectangular magnetic field pulses on 45Ca2+ uptake in resting and mitogen‐treated rat thymic lymphocytes was evaluated. A 30‐min, non‐thermal exposure to the pulsed magnetic field (B
peak = 6.5 mT, E
max = 0.69 mV/cm, J
max = 2.6 μA/cm2) reduced Concanavalin A‐induced 45Ca2+ uptake by 45%. It was observed that (i) the induction of the 3‐Hz field response dependend on Ca2+ signal transduction activation; (ii) the response direction (stimulation or inhibition) depended on the level of lymphocyte mitogen responsiveness, and (iii) the field response magnitude increased with increasing magnetic field flux densities (B
peak = 0, 1.6, 6.5 and 28 mT). Our results demonstrate field effects at B
max nearly 104 greater than that of the average human environment for low‐frequency magnetic fields and they are consistent with the independent results from other 3‐Hz pulsed magnetic field studies with lymphocytes.
The glycine residue of ATP regulatory module in receptor guanylate cyclases that is essential in natriuretic factor signaling
Authors: Duda Teresa , Goraczniak Rafal M. , Sharma Rameshwar K.
Published: July 2002

Atrial natriuretic factor (ANF) and C‐type natriuretic peptide (CNP)‐activated guanylate cyclases are single‐chain transmembrane‐spanning proteins, containing both ligand binding and catalytic activities. In both proteins, ligand binding to the extracellular receptor domain activates the cytosolic catalytic domain, generating the second messenger cyclic GMP. Obligatory in this activation process is an ATP‐dependent step. ATP directly binds to a defined ATP‐regulatory module (ARM) sequence motif in the cyclases and through ARM bridges the events of ligand binding and signal transduction. These ARM sequence motifs are respectively represented by Gly503‐Xa‐Gly505‐Xa‐Xa‐Xa‐Gly509 and Gly499‐Xa‐Xa‐Xa‐Gly503 in the case of ANF receptor guanylate cyclase (ANF‐RGC) and CNP receptor guanylate cyclase (CNP‐RGC). Through genetic remodeling techniques, we now show that ARM‐Gly505 in ANF‐RGC and the corresponding ARM‐Gly499 in CNP‐RGC are critical for ANF and CNP signaling, and other ARM‐Gly residues have minimal effect in the respective signaling processes.
Distinct expression of two Drosophila homologs of fibroblast growth factor receptors in imaginai discs
Authors: Emori Yasufumi , Saigo Kaoru
Published: July 2002

The expression of two Drosophila homologs of FGF receptors (DRF1 and DRF2) in imaginal discs was studied. DFR1 mRNA was observed in several imaginal discs, whereas DFR2 mRNA was not detected. DFR1 expression in the wing and leg discs took place in probable myoblasts in a pattern similar to that of twist, a mesodermal gene. The mRNA was also detected in the morphogenetic furrow and its posterior region of the eye disc and around the proliferation center of the brain. These results suggest that DFR1 is involved in the development of mesodermal and neuronal cells constituting the adult body.
Suppression of neuronal potassium A‐current by arachidonic acid
Authors: Villarroel Alvaro
Published: July 2002

The effect of arachidonic acid on the A current (I
A) has been studied in dissociated bullfrog neurons under whole‐cell voltage‐damp conditions. Arachidonic acid reduced I
A in a dose‐dependent and reversible manner without a shift in the prepulse inactivation voltage‐current relation. 1.75 μM inhibited i
A by 50%, and higher concentrations caused a total suppression. In addition, arachidonic add increased the M‐current (I
M), a different potassium current that does not inactivate. Neither indomethacin nor nordihydroguaiaretic acid, cyclooxygenase and lipoxygenase inhibitors respectively, prevented I
A reduction. In contrast, nordihydroguaiaretic acid prevented I
M enhancement. Eicosatetraynoic acid, an arachidonic acid analog that cannot be metabolized, also reduced I
A. These results suggest that arachidonic acid metabolism is not required to suppress I
A
A plasma‐membrane progesterone receptor in human sperm is switched on by increasing intracellular free calcium
Authors: Mendoza C. , Tesarik J.
Published: July 2002

Recent studies have revealed the presence of an atypical progesterone receptor on the plasma membrane of a relatively small subpopulation of human sperm cells. In this study, we show that the receptor is present in many cells in a latent form and that the receptor function in these cells is switched on asynchronously by increasing intracellular free Ca2+ levels. Under physiological conditions, this process occurs during sperm capacitation, but it can be mimicked by a short sperm exposure to the Ca2+‐mobilizing agent thapsigargin. These observations suggest a role of Ca2+ as a modulator of nongenomic steroid effects on the cell surface.
Identification of mitogen‐activated protein kinase phosphorylation sequences in mammalian h‐Caldesmon
Authors: Adam Leonard P. , Hathaway David R.
Published: July 2002

h‐Caldesmon in vascular smooth muscle is phosphorylated in response to pharmacologie stimulation. Although many kinases phosphorylate h‐caldesmon, in vitro, the responsible kinase in intact tissue is unknown. The sites of phosphorylation in caldesmon from intact canine aortas have recently been identified and are consensus sequences for a proline‐directed protein kinase. In this study, we investigated the phosphorylation of h‐caldesmon by mitogen‐activated protein kinase (MAPK). Purified, recombinant MAPK phosphorylated porcine stomach h‐caldesmon to a stoichiometry approaching 2 mol phosphate/mol protein. Phosphorylated h‐caldesmon was subjected to proteolysis and the phosphopeptides were purified by high performance liquid chromatography. Two major phosphopeptides were identified and sequenced. These two peptides, VTS ∗ PTKV and S ∗ PAPK, were identical to the sequences of the sites phosphorylated in intact tissue. Antibodies to several enzymes implicated in the cascade of activation of MAPK were used to evaluate vascular smooth muscle by Western blotting. All components were found to be present. These data suggest that MAPK can function as a ‘caldesmon kinase’ in vascular smooth muscle.