Search PubMed⌕ Search

Biomedical subjects

K E Bernstein

Publications and source records attributed to K E Bernstein.

At least 55 records · Page 3Linked to original sources
PubMed1996

Angiotensin II-induced tyrosine phosphorylation in mesangial and vascular smooth muscle cells.

1. Angiotensin II (AngII)-induced, activation of phospholipase C (PLC) and Ca2+-dependent Cl- channels is an important signal transduction pathway for the regulation of vascular smooth muscle cell (VSMC) and glomerular mesangial cell contraction and growth. While AT receptors are traditionally thought to be G-protein coupled to the beta isoform of PLC, recent evidence suggests that in some tissues AT receptors may also activate the PLC-gamma isoform via tyrosine phosphorylation. 2. By western analysis, we identified PLC-gamma1 in the above cell types. We found that within 3 min of exposure to 10(-7) mol/L AngII, tyrosine phosphorylation of PLC-gamma1 was observed; however, peak response (>3-fold increase) occurred within 0.5 min. In addition, pre-incubation of these cells with the tyrosine kinase inhibitor genistein blocked the tyrosine phosphorylation of PLC-gamma1 by AngII. In contrast, preincubation with the tyrosine phosphatase inhibitor sodium vanadate increased the levels of tyrosine phosphorylation of PLC-gamma1. Similar results were found when intracellular levels of 1,4,5-IP3 were measured after AngII exposure. 3. By using patch clamp techniques on cultured rat mesangial cells exposed to AngII, we found that the release of 1,4,5-IP3-sensitive intracellular Ca2+ stores stimulated low conductance Cl- channels. Preincubation with genistein, abolished the usual 10-fold increase in Cl- channel activity observed with AngII. 4. Therefore, we conclude that in VSMC and glomerular mesangial cells (i) AngII transiently stimulates PLC activity via tyrosine phosphorylation of the gamma1 isoenzyme, (ii) tyrosine phosphorylation of PLC-gamma1 and production of 1,4,5-IP3 in response to AngII is dramatically inhibited by tyrosine kinase inhibition and stimulated by tyrosine phosphatase inhibition, (iii) activation of Ca2+-dependent Cl- channels by AngII-induced release of 1,4,5-IP3-dependent intracellular Ca2+ stores is also abolished by tyrosine kinase inhibition. In summary, this AngII-induced signal transduction cascade provides a possible mechanism for both the contractile and growth-stimulating effects of AngII on VSMC and glomerular mesangial cells.

Angiotensin II↗
PubMed1995

Electroporation of pp60c-src antibodies inhibits the angiotensin II activation of phospholipase C-gamma 1 in rat aortic smooth muscle cells.

Our previous study has shown that angiotensin II induces the rapid tyrosine phosphorylation and activation of phospholipase C-gamma 1 in cultured rat aortic smooth muscle (RASM) cells (Marrero, M.B., Paxton, W.G., Duff, J. L., Berk, B. C., and Bernstein, K. E. (1994) J. Biol. Chem, 269, 10935-10939). This signaling pathway is initiated by ligand binding to the AT1 receptor, a cell surface G protein-coupled receptor. Antibodies to pp60c-src were introduced into RASM cells by electroporation. Angiotensin II-stimulated tyrosine phosphorylation of phospholipase C-gamma 1 was eliminated by the anti-pp60c-src antibodies but not by anti-mouse IgG or bovine serum albumin. Angiotensin II also induced the rapid tyrosine phosphorylation of pp120, a known pp60c-src kinase substrate, and this phosphorylation was also specifically inhibited by anti-pp60c-src antibodies. Electroporation of RASM cells with anti-pp60c-src antibodies had no effect on platelet-derived growth factor-stimulated tyrosine phosphorylation of PLC-gamma 1. Anti-pp60c-src also reduced the angiotensin II-stimulated inositol 1,4,5-trisphosphate production by 78%, while it had no effect on the platelet-derived growth factor-stimulated inositol 1,4,5-trisphosphate production. These data provide the first evidence for a direct involvement of pp60c-src kinase in angiotensin II-mediated PLC-gamma 1 phosphorylation and activation. Furthermore, it also describes a pathway in which a seven-transmembrane receptor can stimulate an intracellular tyrosine kinase.

Angiotensin II↗
PubMed1995

Direct stimulation of Jak/STAT pathway by the angiotensin II AT1 receptor.

The peptide angiotensin II is the effector molecule of the reninangiotensin system. All the haemodynamic effects of angiotensin II, including vasoconstriction and adrenal aldosterone release, are mediated through a single class of cell-surface receptors known as AT1 (refs 1, 2). These receptors contain the structural features of the G-protein-coupled receptor superfamily. We show here that angiotensin II induces the rapid phosphorylation of tyrosine in the intracellular kinases Jak2 and Tyk2 in rat aortic smooth-muscle cells and that this phosphorylation is associated with increased activity of Jak2. The Jak family substrates STAT1 and STAT2 (for signal transducers and activators of transcription) are rapidly tyrosine-phosphorylated in response to angiotensin II. We also find that Jak2 co-precipitates with the AT1 receptor, indicating that G-protein-coupled receptors may be able to signal through the intracellular phosphorylation pathways used by cytokine receptors.

Angiotensin II↗
PubMed1995

Identification of two positive transcriptional elements within the 91-base pair promoter for mouse testis angiotensin converting enzyme (testis ACE).

Testis angiotensin-converting enzyme (testis ACE) is an isozyme of ACE only expressed by male germ cells during spermiogenesis. It is the result of a strong sperm-specific promoter found within the 12th intron of the somatic ACE gene. Previous studies have localized the boundaries of the mouse testis ACE promoter as being from -91 to -9, relative to the transcriptional start site, and have suggested two important DNA regulatory elements starting at positions -55 and -32. DNA constructs were made in which these motifs were either eliminated or substituted. Each construct was tested for its ability to promote transcription in vitro, using a rat testis nuclear extract. Disruption of either motif reduced in vitro transcription to about 30% of control levels, while mutations of both elements abolished transcription. Two sites were selected inside each motif and altered by point mutation. Each of four constructs, containing a mutation at -51, -48, -30, or -28, transcribed at 29% or less the efficiency of the parent construct. The DNA element at -55, TGAGGTCA, is homologous to a consensus cyclic AMP response element. The motif at -32, TCTTAT, is located at a position analogous to a TATA box. Substitution of the -32 motif with a consensus TATA box sequence, TATAAA, stimulated transcriptional activity about 3-fold. As measured by gel mobility shift, oligonucleotides encompassing the -32 motif and the consensus TATA box formed different DNA-protein complexes. However, the -32 motif oligonucleotide was recognized by nuclear proteins prepared from either liver or testis nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

BULLET: a computer simulation of shotgun DNA sequencing.

BULLET is a computer program that simulates shotgun sequencing of DNA. The program has been used to simulate the sequencing of DNA fragments from 2 to 30 kb. To obtain 80% single or double-stranded sequence, a doubling of the DNA fragment size necessitates twice the number of sequencing reactions. However, a similar linear relationship does not apply to the determination of 100% single or double-stranded sequence. Data from BULLET were used to derive simple linear formulae that estimate the number of sequencing reactions necessary to partially sequence a fragment of DNA irrespective of size. When 80% of the double-stranded sequence has been determined, approximately 98% of single-stranded sequence will be known. This is a reasonable point to change to a more directed strategy of DNA sequencing.

Algorithms↗
PubMed1995

Glucocorticoids induce angiotensin-converting enzyme expression in vascular smooth muscle.

Angiotensin-converting enzyme (ACE) activity plays a central role in vessel growth and remodeling as shown by the fact that ACE inhibitors reduce neointimal proliferation after rat carotid injury. To investigate the mechanisms that regulate smooth muscle cell ACE expression, we studied the effects of steroids on ACE activity and mRNA in cultured rat aortic smooth muscle cells. ACE activity was present at low levels independent of growth state. In response to the glucocorticoid dexamethasone (100 nmol/L for 72 hours), ACE activity (hydrolysis of [3H]benzoyl-Phe-Ala-Pro) increased 10.1 +/- 3.1-fold. The increase in activity occurred within 12 hours and peaked after 72 hours of treatment. The increase in ACE activity was specific for glucocorticoids and paralleled their potency (dexamethasone > hydrocortisone = prednisolone). Dexamethasone increased the steady-state level of ACE mRNA in a concentration-dependent manner (21.4 +/- 0.4-fold at 100 nmol/L for 72 hours). Dexamethasone stimulation of ACE expression appeared to be due to both increased transcription and stabilization of ACE enzyme mRNA. This was suggested by the finding that dexamethasone stimulated nuclear run-on expression of ACE mRNA by only threefold, in contrast to the 21-fold increase in steady-state mRNA. These findings establish that ACE is a dynamically regulated enzyme in rat aortic smooth muscle cells. In addition, the present findings suggest an important role for stress steroids in the vascular response to injury in vivo.

Animals↗
PubMed1995

Angiotensin II activates pp60c-src in vascular smooth muscle cells.

The angiotensin II type-1 (AT1) receptor, a G protein-coupled receptor, lacks intrinsic kinase activity. However, recent data show that angiotensin II (Ang II) stimulates tyrosine phosphorylation of phospholipase C-gamma 1 (PLC-gamma 1), Stat91 (one of the signal transducers and activators of transcription), and paxillin in vascular smooth muscle cells. The tyrosine kinases responsible for these phosphorylation events are unknown. Src family kinases have been shown to phosphorylate PLC-gamma 1 and to be activated by G protein-coupled receptors. We hypothesized that pp60c-src associates with the AT1 receptor and is activated after Ang II stimulation of smooth muscle cells. We immunoprecipitated pp60c-src from Ang II-stimulated vascular smooth muscle cells and measured pp60c-src activity by autophosphorylation and by phosphorylation of enolase. Both assays demonstrated an approximately threefold increase in pp60c-src activity within 1 minute. A similar increase in Ang II-stimulated pp60c-src activity was observed in Chinese hamster ovary cells transfected with the AT1 receptor but not in untransfected cells. These data are the first to show that pp60c-src is activated by Ang II. To determine if pp60c-src associated with the AT1 receptor, the AT1 receptor was immunoprecipitated (with two different antibodies), and Western blots were performed with two different anti-pp60c-src antibodies. No pp60c-src was detected. In addition, direct interaction between the AT1 receptor and pp60c-src could not be demonstrated by using a glutathione S-transferase (GST)-AT1 fusion protein to bind proteins from cell lysates stimulated by Ang II.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II↗
PubMed1995

Fibroblast growth factor stimulates angiotensin converting enzyme expression in vascular smooth muscle cells. Possible mediator of the response to vascular injury.

Angiotensin converting enzyme (ACE) activity contributes to the vascular response to injury because ACE inhibition limits neointima formation in rat carotid arteries after balloon injury. To investigate the mechanisms by which ACE may contribute to vascular smooth muscle cell (VSMC) proliferation, we studied expression of ACE in vivo after injury and in vitro after growth factor stimulation. ACE activity 14 d after injury was increased 3.6-fold in the injured vessel. ACE expression, measured by immunohistochemistry, became apparent at 7 d in the neointima and at 14 d was primarily in the most luminal neointimal cells. To characterize hormones that induce ACE in vivo, cultured VSMC were exposed to steroids and growth factors. Among steroids, only glucocorticoids stimulated ACE expression with an 8.0 +/- 2.1-fold increase in activity and a 6.5-fold increase in mRNA (30 nM dexamethasone for 72 h). Among growth factors tested, only fibroblast growth factor (FGF) stimulated ACE expression (4.2 +/- 0.7-fold increase in activity and 1.6-fold increase in mRNA in response to 10 ng/ml FGF for 24 h). Dexamethasone and FGF were synergistic at the indicated concentrations inducing 50.6 +/- 12.4-fold and 32.5-fold increases in activity and mRNA expression, respectively. In addition, when porcine iliac arteries were transfected with recombinant FGF-1 (in the absence of injury), ACE expression increased in neointimal VSMC, to the same extent as injured, nontransfected arteries. The data suggest a temporal sequence for the response to injury in which FGF induces ACE, ACE generates angiotensin II, and angiotensin II stimulates VSMC growth in concert with FGF.

Animals↗
PubMed1995

Angiotensin II signal transduction and the mitogen-activated protein kinase pathway.

In this review, the angiotensin-II-mediated signal transduction pathways involved in vascular smooth muscle cell growth are discussed. Classical pathways involving phospholipase C and protein kinase C, as well as the mitogen-activated protein kinase pathway, are common signal transduction pathways activated by a variety of growth factors to stimulate cell growth. Besides its vasoconstrictor activity, angiotensin II stimulates hypertrophy of vascular smooth muscle cells and is involved in neointimal proliferation following balloon angioplasty. Understanding angiotensin-II-stimulated signaling events, as well as the crosstalk among signaling pathways, may form the basis for the development of new therapies for hypertension and restenosis.

Angiotensin II↗
PubMed1994

Chicken lacks the testis specific isozyme of angiotensin converting enzyme found in mammals.

Chicken angiotensin converting enzyme (ACE) cDNA was cloned based on homology to the mouse ACE sequence. The chicken ACE protein is highly homologous to the somatic isozyme of ACE found in human, mouse, bovine, and rabbit. Like the mammalian somatic forms, the chicken enzyme consists of two putative zinc binding sites at the center of two homologous domains. All known functional residues are absolutely conserved. Unlike the mammals, no evidence for a single domain, testis specific form of ACE was found in the chicken testis by either Northern blot or enzyme assay. This result is unexpected since the adult mammalian testis expresses one of the highest tissue levels of ACE.

Amino Acid Sequence↗
PubMed1994

The angiotensin II AT1 receptor is tyrosine and serine phosphorylated and can serve as a substrate for the src family of tyrosine kinases.

Angiotensin II AT1 receptor signal transduction has recently been shown to function through the phospholipase C isozyme, PLC-gamma. Since PLC-gamma is known to interact with phosphotyrosine containing proteins through SH2 domains, we examined the phosphorylation state of the AT1 receptor. Immunoprecipitation of the [32P] labeled AT1 receptor from rat aortic smooth muscle cells followed by alkali hydrolysis demonstrated the presence of tyrosine phosphorylation. Phosphoamino acid analysis of the excised bands demonstrated the presence of phosphoserine and phosphotyrosine residues. A fusion protein comprising the intracellular tail of the AT1 receptor was used to screen for candidate kinases, and the src kinase family displayed high activity. In summary, this study shows that the AT1 receptor is serine and tyrosine phosphorylated in vivo and suggests that a soluble kinase related to the src family may be responsible for the tyrosine phosphorylation.

Angiotensin II↗
PubMed1994

Angiotensin II stimulates tyrosine phosphorylation of phospholipase C-gamma 1 in vascular smooth muscle cells.

The receptor for angiotensin II (Ang II) has recently been cloned; it is a receptor with seven transmembrane spanning domains that stimulates phosphoinositide hydrolysis upon ligand binding. The physiologic effects of Ang II are important in the regulation of vascular function. In this study, we examined the ability of Ang II to regulate the enzymatic activity of phospholipase C (PLC) in rat aortic vascular smooth muscle cells (VSMC). In cultured VSMC, PLC-gamma 1 and PLC-delta 1 isozymes, but not PLC-beta 1, were identified by Western analysis. Ang II (10(-7) M)-stimulated PLC-gamma 1 phosphotyrosine phosphorylation with a maximum increase of 4.5-fold at 0.5 min. This followed the same time course as the Ang II-stimulated increase in inositol 1,4,5-trisphosphate (1,4,5-IP3) levels. 1,4,5-IP3 formation was inhibited 75% by the tyrosine kinase inhibitor genistein (120 microM). Several growth factor receptors, such as the platelet-derived growth factor (PDGF) receptor are themselves tyrosine kinases and have been shown to phosphorylate PLC-gamma 1 and increase intracellular Ca2+ concentrations. The time course for PLC-gamma 1 phosphorylation, IP3 formation, and Ca2+ mobilization by PDGF differed from Ang II in VSMC. The kinetics of the PDGF effects were slower in onset and more prolonged than those of Ang II. In summary, these findings show that Ang II stimulates VSMC phosphoinositide hydrolysis in association with tyrosine phosphorylation of PLC-gamma 1 and support the concept that Ang II-stimulated tyrosine phosphorylation is responsible for early signal transduction events.

Angiotensin II↗
PubMed1993

Angiotensin II induces 3CH134, a protein-tyrosine phosphatase, in vascular smooth muscle cells.

Angiotensin II is a potent growth factor for vascular smooth muscle cells and shares many signal transduction mechanisms with mitogens, including stimulation of mitogen-activated protein (MAP) kinases and protein tyrosine phosphorylation. Regulation of tyrosine phosphorylation involves both protein-tyrosine kinases and protein-tyrosine phosphatases (PTPases). To investigate the role of PTPases in angiotensin II-mediated events, we studied the expression of a transcriptionally regulated PTPase, 3CH134, which has selective activity toward MAP kinase. Angiotensin II rapidly induced 3CH134 mRNA (30 min maximum) in a concentration-dependent manner (100 nM maximum). Platelet-derived growth factor, alpha-thrombin, hydrogen peroxide, phorbol 12-myristate 13-acetate, and ionomycin also induced 3CH134 but to levels lower than angiotensin II. Induction of 3CH134 by angiotensin II was partially inhibited after down-regulating protein kinase C but was fully inhibited after chelating intracellular Ca2+. Treatment with both phorbol 12-myristate 13-acetate and ionomycin induced 3CH134 mRNA to levels seen with angiotensin II, indicating that Ca2+ mobilization and protein kinase C activation can act synergistically to induce 3CH134. Angiotensin II stimulated 3CH134 protein synthesis after 1 h as measured by immunoprecipitation of 3CH134 from [35S]methionine-labeled cells using affinity-purified antibodies. These results establish 3CH134 as a dynamically regulated, immediate early gene in vascular smooth muscle cells and suggest a role for PTPases in regulating angiotensin II-stimulated events mediated by MAP kinases and tyrosine kinases.

Angiotensin II↗
PubMed1993

Sequence of a cDNA encoding dog insulin-like growth factor I.

Polymerase chain reaction amplification of a cDNA derived from dog left ventricular myocardium, using primers specific for rat insulin-like growth factor I (IGFI), exons 3 and 6, yielded the dog clone, IGFI5.1. This clone includes the signal peptide sequence, the entire coding sequence for mature dog IGFI and the C-terminal extension sequence. By analogy with the organization of the rat and human IGFI genes which encode two extension peptides, we have termed this cDNA, dog IGFIa. The deduced amino acid sequence of mature dog IGFI is identical to that of human IGFI.

Animals↗
PubMed1993

Mutation of Asp74 of the rat angiotensin II receptor confers changes in antagonist affinities and abolishes G-protein coupling.

Aspartic acid in the second transmembrane domain is a highly conserved amino acid among the G protein-coupled receptors and is functionally important for agonist binding and G-protein coupling in beta 2-adrenergic and luteinizing hormone receptors. To determine whether this aspartic acid is also involved in the function of the rat vascular angiotensin II receptor subtype 1 (AT1a), Asp74 was replaced either by asparagine or by glutamic acid. When expressed in CHO cells, the two mutants and the wild-type receptor displayed similarly high affinities for the agonist [Sar1, Tyr(125I)4]angiotensin II [where Sar is sarcosine and Tyr(125I) is monoiodinated tyrosine] and the other agonists: ([Sar1]angiotensin II > angiotensin II > angiotensin III >> angiotensin I). However, the Asn74 mutant shows striking differences in its affinity for some antagonists when compared with the wild-type receptor: the affinity for DUP753 was decreased 10-fold, whereas it was increased 6-fold for [Sar1,Ala8]angiotensin II and 20-fold for CGP42112A. These pharmacological changes were associated with a major defect in transmembrane signaling, since angiotensin II was unable to stimulate inositol phosphate production and increase cytosolic Ca2+ concentration through the two mutated receptors, whereas a clear dose-dependent stimulation was observed in cells expressing the wild-type receptor. Angiotensin II was able to promote DNA synthesis through the wild type but not through the mutated receptors. These data indicate that the conserved Asp74 residue of the AT1a receptor is important for the binding of angiotensin II antagonists and is essential for the transmembrane signaling cascade.

Amino Acid Sequence↗
PubMed1993

Structure and regulated expression of angiotensin-converting enzyme and the receptor for angiotensin II.

The renin-angiotensin system maintains a homeostasis of blood pressure and blood volume. One component of this system is angiotensin-converting enzyme (ACE). There are two isozymes of ACE. The protein produced by vascular endothelium is termed "somatic ACE" and is regulated as a function of the growth state of these cells in vitro. The second isozyme, "testis ACE," is only produced by developing spermatozoa. The two ACE isozymes are the result of two distinct promoter regions within the ACE gene. Angiotensin II binds to specific receptors on the surface of cells. We have isolated cDNA encoding the AT1 subtype of receptor. This subtype is responsible for the hemodynamic consequences of angiotensin.

Animals↗
PubMed1993

The biology of angiotensin II receptors.

Angiotensin II is very important in the regulation of blood pressure. This small peptide binds to cell surface receptors, initiating a wide diversity of physiologic responses. There are two major subtypes of angiotensin II receptors referred to as AT1 and AT2. In this article we describe the cloning and the biochemical characterization of the AT1 receptor. Antibodies against this receptor have been used to define its tissue distribution. The AT1 receptor is a member of the seven transmembrane spanning class of receptors. It initiates a complex series of signaling events, including activation of membrane phospholipases and intracellular kinases. In the human a single AT1 receptor protein mediates virtually all the effects of angiotensin II, suggesting that tissue specificity of angiotensin II must be due to organ-specific intracellular signaling.

Amino Acid Sequence↗