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Biomedical subjects

A Askari

Publications and source records attributed to A Askari.

At least 19 recordsLinked to original sources
PubMed2001

Role of protein kinase C in the signal pathways that link Na+/K+-ATPase to ERK1/2.

We have shown before that Na(+)/K(+)-ATPase acts as a signal transducer, through protein-protein interactions, in addition to being an ion pump. Interaction of ouabain with the enzyme of the intact cells causes activation of Src, transactivation of EGFR, and activation of the Ras/ERK1/2 cascade. To determine the role of protein kinase C (PKC) in this pathway, neonatal rat cardiac myocytes were exposed to ouabain and assayed for translocation/activation of PKC from cytosolic to particulate fractions. Ouabain caused rapid and sustained stimulation of this translocation, evidenced by the assay of Ca(2+)-dependent and Ca(2+)-independent PKC activities and by the immunoblot analysis of the alpha, delta, and epsilon isoforms of PKC. Dose-dependent stimulation of PKC translocation by ouabain (1-100 microm) was accompanied by no more than 50% inhibition of Na(+)/K(+)-ATPase and doubling of [Ca(2+)](i), changes that do not affect myocyte viability and are known to be associated with positive inotropic, but not toxic, effects of ouabain in rat cardiac ventricles. Ouabain-induced activation of ERK1/2 was blocked by PKC inhibitors calphostin C and chelerythrine. An inhibitor of phosphoinositide turnover in myocytes also antagonized ouabain-induced PKC translocation and ERK1/2 activation. These and previous findings indicate that ouabain-induced activation of PKC and Ras, each linked to Na(+)/K(+)-ATPase through Src/EGFR, are both required for the activation of ERK1/2. Ouabain-induced PKC translocation and ERK1/2 activation were dependent on the presence of Ca(2+) in the medium, suggesting that the signal-transducing and ion-pumping functions of Na(+)/K(+)-ATPase cooperate in activation of these protein kinases and the resulting regulation of contractility and growth of the cardiac myocyte.

Animals↗
PubMed2001

Downregulation of cardiac myocyte Na(+)-K(+)-ATPase by adenovirus-mediated expression of an alpha-subunit fragment.

Cultured rat cardiac myocytes and A7r5 cells were transfected with an adenoviral vector used earlier for in vivo expression of functional alpha(2)-isoform of the catalytic subunit of rat Na(+)-K(+)-ATPase. Expressions of truncated forms of alpha(2), but little or no intact alpha(2), were detected, suggesting the rapid degradation of alpha(2) in these cultured cells. In neonatal myocytes normally containing the alpha(1)- and the alpha(3)-isoforms, expression of the alpha(2)-fragment led to 1) a significant decrease in the level of endogenous alpha(1)-protein and a modest decrease in alpha(3)-protein, 2) decreases in mRNAs of alpha(1) and alpha(3), 3) decrease in Na(+)-K(+)-ATPase function measured as ouabain-sensitive Rb(+) uptake, 4) increase in intracellular Ca(2+) concentration similar to that induced by ouabain, and 5) eventual loss of cell viability. These findings indicate that the alpha(2)-fragment downregulates endogenous Na(+)-K(+)- ATPase most likely by dominant negative interference either with folding and/or assembly of the predominant housekeeping alpha(1)-isoform or with signal transducing function of the enzyme. Demonstration of rise in intracellular Ca(2+) resulting from alpha(1)-downregulation 1) does not support the previously suggested special roles of less abundant alpha(2)- and alpha(3)-isoforms in the regulation of cardiac Ca(2+), 2) lends indirect support to proposals that observed decrease in total Na(+)-K(+)-ATPase of the failing heart may be a mechanism to compensate for impaired cardiac contractility, and 3) suggests the potential therapeutic utility of dominant negative inhibition of Na(+)-K(+)-ATPase.

Adenoviridae↗
PubMed2000

Ouabain interaction with cardiac Na+/K+-ATPase initiates signal cascades independent of changes in intracellular Na+ and Ca2+ concentrations.

We have shown previously that partial inhibition of the cardiac myocyte Na(+)/K(+)-ATPase activates signal pathways that regulate myocyte growth and growth-related genes and that increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) and reactive oxygen species (ROS) are two essential second messengers within these pathways. The aim of this work was to explore the relation between [Ca(2+)](i) and ROS. When myocytes were in a Ca(2+)-free medium, ouabain caused no change in [Ca(2+)](i), but it increased ROS as it did when the cells were in a Ca(2+)-containing medium. Ouabain-induced increase in ROS also occurred under conditions where there was little or no change in [Na(+)](i). Exposure of myocytes in Ca(2+)-free medium to monensin did not increase ROS. Increase in protein tyrosine phosphorylation, an early event induced by ouabain, was also independent of changes in [Ca(2+)](i) and [Na(+)](i). Ouabain-induced generation of ROS in myocytes was antagonized by genistein, a dominant negative Ras, and myxothiazol/diphenyleneiodonium, indicating a mitochondrial origin for the Ras-dependent ROS generation. These findings, along with our previous data, indicate that increases in [Ca(2+)](i) and ROS in cardiac myocytes are induced by two parallel pathways initiated at the plasma membrane: One being the ouabain-altered transient interactions of a fraction of the Na(+)/K(+)-ATPase with neighboring proteins (Src, growth factor receptors, adaptor proteins, and Ras) leading to ROS generation, and the other, inhibition of the transport function of another fraction of the Na(+)/K(+)-ATPase leading to rise in [Ca(2+)](i). Evidently, the gene regulatory effects of ouabain in cardiac myocytes require the downstream collaborations of ROS and [Ca(2+)](i).

Animals↗
PubMed2000

Involvement of Src and epidermal growth factor receptor in the signal-transducing function of Na+/K+-ATPase.

Nontoxic concentrations of ouabain, causing partial inhibition of the cardiac myocyte Na(+)/K(+)-ATPase, induce hypertrophy and several growth-related genes through signal pathways that include the activation of Ras and p42/44 mitogen-activated protein kinase (MAPK). The aim of this work was to examine the ouabain-induced events upstream of the Ras/MAPK cascade. Treatment of myocytes with genistein antagonized ouabain-induced activation of the MAPK, suggesting that protein tyrosine phosphorylation has a role. Tyrosine phosphorylation of several myocyte proteins was increased rapidly upon cell exposure to ouabain. Lowering of extracellular K(+) had a similar ouabain-like effect. Ouabain also increased protein tyrosine phosphorylation in A7r5, HeLa, and L929 cells. In cardiac myocytes and A7r5 cells, herbimycin A antagonized the ouabain-induced increase in protein tyrosine phosphorylation and MAPK activation. In both cell types, ouabain stimulated Src kinase activity, Src translocation to the Triton-insoluble fraction, Src association with the epidermal growth factor receptor, and the tyrosine phosphorylation of this receptor on site(s) other than its major autophosphorylation site, Tyr(1173). The findings suggest that (a) the ouabain-induced activation of Src and the Src-induced phosphorylation of the growth factor receptor provide the scaffolding for the recruitment of adaptor proteins and Ras and the activation of Ras/MAPK cascade; and (b) the activation of such pathways may be a common feature of the signal-transducing function of Na(+)/K(+)-ATPase in most cells.

Amino Acid Sequence↗
PubMed2000

Evaluation of pathergy test in North Jordan.

The positivity of pathergy's test in Behçet disease varies throughout the world. We tried to evaluate its diagnostic significance in known Behçet patients in the region of North Jordan. Twenty-two patients were tested, readings were taken after 48 hours. The patients were not tested previously and a comparison between the subcutaneous injection of 0.1 ml normal saline and skin puncture was recorded. The development of a skin eruption (papule, nodule, pustule) were observed and described. The percentage of positivity was 20% in the studied population. The patients tested who were not taking medication at the time of the procedure are more likely to illustrate a positive Pathergy reaction.

Adult↗
PubMed2000

Ankylosing spondylitis in north Jordan: descriptive and analytical study.

OBJECTIVE: A study of ankylosing spondylitis in Jordan, which has been under investigated in this region. METHODS: Twenty two patients were studied according to standard methodology during a period of four years. Information on HLA, the presence of uveitis, cardiac disease, and peripheral arthritis was recorded. Other variables such as age, sex, employment, and level of disability were also recorded. RESULTS: The results reflected the characteristics of the illness and the impact of the disease on the patients and their quality of life and were consistent with the findings of other workers in the region.

Adult↗
PubMed1999

Intracellular reactive oxygen species mediate the linkage of Na+/K+-ATPase to hypertrophy and its marker genes in cardiac myocytes.

We showed before that in cardiac myocytes partial inhibition of Na+/K+-ATPase by nontoxic concentrations of ouabain causes hypertrophy and transcriptional regulations of growth-related marker genes through multiple Ca2+-dependent signal pathways many of which involve Ras and p42/44 mitogen-activated protein kinases. The aim of this work was to explore the roles of intracellular reactive oxygen species (ROS) in these ouabain-initiated pathways. Ouabain caused a rapid generation of ROS within the myocytes that was prevented by preexposure of cells to N-acetylcysteine (NAC) or vitamin E. These antioxidants also blocked or attenuated the following actions of ouabain: inductions of the genes of skeletal alpha-actin and atrial natriuretic factor, repression of the gene of the alpha3-subunit of Na+/K+-ATPase, activation of mitogen-activated protein kinases, activation of Ras-dependent protein synthesis, and activation of transcription factor NF-kappaB. Induction of c-fos and activation of AP-1 by ouabain were not sensitive to NAC. Ouabain-induced inhibition of active Rb+ uptake through Na+/K+-ATPase and the resulting rise in intracellular Ca2+ were also not prevented by NAC. A phorbol ester that also causes myocyte hypertrophy did not increase ROS generation, and its effects on marker genes and protein synthesis were not affected by NAC. We conclude the following: (a) ROS are essential second messengers within some but not all signal pathways that are activated by the effect of ouabain on Na+/K+-ATPase; (b) the ROS-dependent pathways are involved in ouabain-induced hypertrophy; (c) increased ROS generation is not a common response of the myocyte to all hypertrophic stimuli; and (d) it may be possible to dissociate the positive inotropic effect of ouabain from its growth-related effects by alteration of the redox state of the cardiac myocyte.

Acetylcysteine↗
PubMed1999

Microscopic polyangiitis presenting as schizophrenia.

Primary vasculitides can affect the central nervous system but the psychiatric manifestations are not well described. The hallmark of the disorder is an acute necrotizing inflammation of the vessel media with fibrinoid necrosis. We report a case of a 31-year-old man who had been treated for schizophrenia but was found to have vasculitides of the microscopic polyangiitis type and who improved on therapy with corticosteroids and immunosuppressive drugs. We would like to draw attention to this rare but important manifestation of vasculitis, and to the importance of its recognition.

Adrenal Cortex Hormones↗
PubMed1999

Sjögren's syndrome: a critical review of clinical management.

Sjögren's syndrome (SS) is greatly under recognized in clinical practice, primarily for 2 reasons: its presentations are variable and often nonspecific and there are still no clear, uniform diagnostic criteria for this clinical entity. The prevalence, natural history, pathogenesis, and clinical taxonomy of SS are still not well understood. Potential criteria include both subjective symptoms and objective criteria such as measurements of salivary and tear flow, minor salivary gland biopsy, and an increasing variety of serological markers. Physicians often fail to appreciate the profound impact of SS on quality of life. Therefore, screening for SS should include questions exploring symptoms in terms of their effect on the patient's daily life. At present, there is no curative treatment for SS. For symptom relief, local treatments (such as artificial tears or oral topical sprays) are limited in their effects, whereas systemic treatment offers the advantage of addressing a wider range of symptoms. Controlled studies show that oral pilocarpine significantly improves sicca symptoms in the eyes, mouth, and other sites. Clinical experience to date suggests it is safe and well tolerated, with no serious adverse effects, tachyphylaxis, or drug to drug interactions of concern. The most frequent adverse effects are sweating, urinary frequency, diarrhea, and other parasympathomimetic effects, but these do not lead to substantial drug withdrawal rates. Patients should be forewarned that subjective improvement may lag behind improvement in objective measures. Because management often spans several specialties, coordination among them is essential. Dental, gynecological, and ophthalmological perspectives on diagnosis and management are discussed; the primary practitioner has the opportunity to play both a coordinating role and a direct role in early diagnosis and treatment.

Combined Modality Therapy↗
PubMed1998

Ouabain-sensitive H,K-ATPase: tissue-specific expression of the mammalian genes encoding the catalytic alpha subunit.

Human ATP1AL1 and corresponding genes of other mammals encode the catalytic alpha subunit of a non-gastric ouabain-sensitive H,K-ATPases, the ion pump presumably involved in maintenance of potassium homeostasis. The tissue specificity of the expression of these genes in different species has not been analyzed in detail. Here we report comparative RT-PCR screening of mouse, rat, rabbit, human, and dog tissues. Significant expression levels were observed in the skin, kidney and distal colon of all species (with the exception of the human colon). Analysis of rat urogenital organs also revealed strong expression in coagulating and preputial glands. Relatively lower expression levels were detected in many other tissues including brain, placenta and lung. In rabbit brain the expression was found to be specific to choroid plexus and cortex. Prominent similarity of tissue-specific expression patterns indicates that animal and human non-gastric H,K-ATPases are indeed products of homologous genes. This is also consistent with the high sequence similarity of non-gastric H,K-ATPases (including partial sequences of hitherto unknown cDNAs for mouse and dog proteins).

Amino Acid Sequence↗
PubMed1998

Multiple signal transduction pathways link Na+/K+-ATPase to growth-related genes in cardiac myocytes. The roles of Ras and mitogen-activated protein kinases.

We showed before that in neonatal rat cardiac myocytes partial inhibition of Na+/K+-ATPase by nontoxic concentrations of ouabain causes hypertrophic growth and transcriptional regulations of genes that are markers of cardiac hypertrophy. In view of the suggested roles of Ras and p42/44 mitogen-activated protein kinases (MAPKs) as key mediators of cardiac hypertrophy, the aim of this work was to explore their roles in ouabain-initiated signal pathways regulating four growth-related genes of these myocytes, i.e. those for c-Fos, skeletal alpha-actin, atrial natriuretic factor, and the alpha3-subunit of Na+/K+-ATPase. Ouabain caused rapid activations of Ras and p42/44 MAPKs; the latter was sustained longer than 90 min. Using high efficiency adenoviral-mediated expression of a dominant-negative Ras mutant, and a specific inhibitor of MAPK kinase (MEK), activation of Ras-Raf-MEK-p42/44 MAPK cascade by ouabain was shown. The effects of the mutant Ras, an inhibitor of Ras farnesylation, and the MEK inhibitor on ouabain-induced changes in mRNAs of the four genes indicated that (a) skeletal alpha-actin induction was dependent on Ras but not on p42/44 MAPKs, (b) alpha3 repression was dependent on the Ras-p42/44 MAPK cascade, and (c) induction of c-fos or atrial natriuretic factor gene occurred partly through the Ras-p42/44 MAPK cascade, and partly through pathways independent of Ras and p42/44 MAPKs. All ouabain effects required extracellular Ca2+, and were attenuated by a Ca2+/calmodulin antagonist or a protein kinase C inhibitor. The findings show that (a) signal pathways linked to sarcolemmal Na+/K+-ATPase share early segments involving Ca2+ and protein kinase C, but diverge into multiple branches only some of which involve Ras, or p42/44 MAPKs, or both; and (b) there are significant differences between this network and the related gene regulatory pathways activated by other hypertrophic stimuli, including those whose responses involve increases in intracellular free Ca2+ through different mechanisms.

Actins↗
PubMed1997

Structural analysis of the products of chymotryptic cleavage of the E1 form of Na,K-ATPase alpha-subunit: identification of the N-terminal fragments containing the transmembrane H1-H2 domain.

Chymotryptic cleavage of the Na,K-ATPase in NaCl medium abolishes ATPase activity and alters other functional parameters. The structure of this modified enzyme is uncertain since only one product of selective proteolysis, the 83-kDa fragment of the alpha-subunit (Ala267-C-terminus) has been identified previously. Here, we applied additional tryptic digestion followed by oxidative cross-linking to identify the products originating from the N-terminal part of the alpha-subunit. These fragments start at Ala72 or Thr74 and contain the transmembrane H1-H2 domain. Formation of cross-linked product between alpha-fragments containing H1-H2 and H7-H10 demonstrate that the structural integrity of the membrane moiety is preserved. We also determined that secondary cleavage of the 83-kDa fragment leads to the formation of C-terminal 48-kDa alpha-fragments with multiple N-termini at Ile582, Ser583, Met584 and Ile585.

Animals↗
PubMed1997

Enzymatic properties of human Na,K-ATPase alpha1beta3 isozyme.

Recent results of a wide-scale human cDNA sequencing project have identified a cDNA which encodes a hitherto unknown human protein sequence exhibiting structural similarities with beta-subunits of the Na,K- and H,K-ATPase family and with the amphibian Na,KATPase beta3-subunit, in particular. In this study the ability of the putative human beta3-subunit to assemble with the human alpha1-subunit in functionally active Na,KATPase was examined using the baculovirus expression system. The recombinant baculovirus simultaneously expressing both alpha1 and beta3 human proteins was produced using the dual-promoter transfer vector p2Bac. The expression of both human proteins in baculovirus-infected Sf-9 cell membranes detected with specific antibodies resulted in the formation of a catalytically competent alpha1beta3 ATPase complex. Characterization of the recombinant ATPase complex involved the analysis of Na+, K+, and ATP dependencies of enzyme activity and its sensitivity toward ouabain. Preparations of HeLa cell membranes containing alpha1beta1 isozyme of human Na,K-ATPase were used as control. The data obtained clearly demonstrated that alpha1beta3 ATPase exhibits enzymatic properties which are characteristic of Na, K-ATPase. The recombinant alpha1beta3 isozyme displayed significantly lower sensitivity to ouabain than native alpha1beta1. These findings indicate that the hitherto unknown alpha1beta3 isozyme of human Na,K-ATPase is likely to exist in vivo, thus suggesting further expansion of human Na,K-ATPase isozyme diversity. The present studies are the first in which heterologous expression has been used for the characterization of an isozyme of human Na, K-ATPase.

Adenosine Triphosphate↗
PubMed1997

Evidence for the existence of two ATP-sensitive Rb+ occlusion pockets within the transmembrane domains of Na+/K+-ATPase.

A trypsin-digested Na+/K+-ATPase that has lost ATPase activity and about half of its protein content retains an essentially intact beta-subunit, the 10 transmembrane domains of the alpha-subunit, and the full capacity to occlude Na+ and Rb+ (a congener of K+). When this preparation was incubated at 37 degrees C in the absence of Rb+, it lost half of its Rb+ occluding capacity and two-thirds of its Na+ occluding capacity. Comparison of the Rb+ occlusion-deocclusion kinetics of the digested enzyme before and after partial inactivation indicated that (a) the affinities of the labile and the stable halves of occluded Rb+ were the same; (b) occlusion and deocclusion rates of the stable pool were lower than those of the labile pool; (c) ATP at a low affinity site (K0.5 = 25-300 microM) increased deocclusion rate in the stable pool and occlusion rate in the labile pool; (d) Na+ increased Rb+ deocclusion rate of the sum of the two pools but not that of the stable pool; and (e) occlusion and deocclusion rates of both pools were decreased by ouabain. These findings suggest that (a) the peptide complex of the digested enzyme contains two distinct but interacting cation occlusion pockets, one occluding two Na+ or one Rb+, and the other occluding one Na+ or one Rb+; (b) this peptide complex that is devoid of the catalytic ATP site retains an allosteric ATP site; and (c) the access channels of the two pockets are regulated differently by ATP but similarly by ouabain. Analyses of the gel electrophoretic patterns of the digested enzyme and the N termini of the appropriate bands showed that inactivation of the labile occlusion pocket was accompanied by 60-70% loss of two alpha-fragments containing H3-H4 and H5-H6 transmembrane domains. This and the previously established interactions among the transmembrane helices of alpha- and beta-subunits suggest that one occlusion pocket is associated with H3-H6 domains and that the other is located within a complex of beta-subunit and two alpha-fragments containing H1-H2 and H7-H10 transmembrane domains.

Animals↗
PubMed1997

Ligand-sensitive interactions among the transmembrane helices of Na+/K+-ATPase.

An extensively trypsin-digested Na+/K+-ATPase, which retains the ability to bind Na+, K+, and ouabain, consists of four fragments of the alpha-subunit that contain all 10 transmembrane alpha domains, and the beta-subunit, a fraction of which is cleaved at Arg142-Gly143. In previous studies, we solubilized this preparation with a detergent and mapped the relative positions of several transmembrane helices of the subunits by chemical cross-linking. To determine if these detected helix-helix proximities were representative of those existing in the bilayer prior to solubilization, we have now done similar studies on the membrane-bound preparation of the same digested enzyme. After oxidative sulfhydryl cross-linking catalyzed by Cu2+-phenanthroline, two prominent products were identified by their mobilities and the analyses of their N termini. One was a dimer of a 11-kDa alpha-fragment containing the H1-H2 helices and a 22-kDa alpha-fragment containing the H7-H10 helices. This dimer seemed to be the same as that obtained in the solubilized preparation. The other product was a trimer of the above two alpha-fragments and that fraction of beta whose extracellular domain was cleaved at Arg142-Gly143. This product was different from a similar one of the solubilized preparation in that the latter contained the predominant fraction of beta without the extracellular cleavage. The cross-linking reactions of the membrane preparation, but not those of the solubilized one, were hindered specifically by Na+, K+, and ouabain. These findings indicate that (a) the H1-H2 transmembrane helices of alpha are adjacent to some of its H7-H10 helices both in solubilized and membrane-bound states, (b) the alignment of the residues of the single transmembrane helix of beta with the interacting H1-H2 and H7-H10 helices of alpha is altered by detergent solubilization and by structural changes in the extracellular domain of beta, and (c) the three-dimensional packing of the interacting transmembrane helices of alpha and beta are regulated by the specific ligands of the enzyme.

Amino Acid Sequence↗