PubMed Health⌕ Search

Biomedical subjects

László Hunyady

Publications and source records attributed to László Hunyady.

At least 19 recordsLinked to original sources
PubMed2007

The role of diacylglycerol lipase in constitutive and angiotensin AT1 receptor-stimulated cannabinoid CB1 receptor activity.

The cannabinoid CB1 receptor (CB1R) is a G protein-coupled receptor, which couples to the Gi/o family of heterotrimeric G proteins. The receptor displays both basal and agonist-induced signaling and internalization. Although basal activity of CB1Rs is attributed to constitutive (agonist-independent) receptor activity, studies in neurons suggested a role of postsynaptic endocannabinoid (eCB) release in the persistent activity of presynaptic CB1Rs. To elucidate the role of eCBs in basal CB1R activity, we have investigated the role of diacylglycerol lipase (DAGL) in this process in Chinese hamster ovary (CHO) cells, which are not targeted specifically with eCBs. Agonist-induced G protein activation was determined by detecting dissociation G protein subunits expressed in CHO cells with bioluminescence resonance energy transfer (BRET), after labeling the alpha and beta subunits with Renilla luciferase and enhanced yellow fluorescent protein (EYFP), respectively. Preincubation of the cells with tetrahydrolipstatin (THL), a known inhibitor of DAGLs, caused inhibition of the basal activity of CB1R. Moreover, preincubation of CHO and cultured hippocampal neurons with THL increased the number of CB1Rs on the cell membrane, which reflects its inhibitory action on CB1R internalization in non-simulated cells. In CHO cells co-expressing CB1R and angiotensin AT1 receptors, angiotensin II-induced Go protein activation that was blocked by both a CB1R antagonist and THL. These data indicate that cell-derived eCB mediators have a general role in the basal activity of CB1Rs in non-neural cells and neurons, and that this mechanism can be stimulated by AT1 receptor activation.

Animals↗
PubMed2006

Menin and its interacting proteins: elucidation of menin function.

The multiple endocrine neoplasia type 1 (MEN1) gene is a tumor suppressor gene encoding a 610 amino acid nuclear protein, menin. Although mutations of the MEN1 gene are responsible for MEN 1 syndrome, the intracellular functions of menin have not been fully elucidated. Recent data suggest that interactions between menin and menin-interacting proteins have a role in physiological regulation of cell growth, control of the cell cycle and genome stability, and are potentially important in bone development and multipotent mesenchymal stem cell differentiation. Loss of these interactions might also contribute to the development of MEN 1 syndrome.

Bone Development↗
PubMed2006

The role of lumican and keratocan genes in persistent subepithelial corneal haze following excimer laser photorefractive keratectomy.

PURPOSE: A retrospective clinical and a genetic study was carried out of severe subepithelial corneal haze occurring after photorefractive keratectomy (PRK). Since this clinical condition resembles the lumican-null mouse phenotype, mutation analysis of lumican and keratocan was carried out to investigate whether germline genetic alterations have an effect on development of severe corneal haze in humans. Corneal thickness, photoablation depth, and severity of persistent corneal haze were also analyzed. In vivo confocal microscopy examination was also performed to study corneal structure and endothelial cells. METHODS: Severity of corneal haze was evaluated by slit-lamp biomicroscopy according to Hanna's scale. Corneal structure and endothelial cell shapes and density were viewed with a scanning confocal microscope. PCR-based mutational analysis was performed using temperature gradient gel electrophoresis (TGGE) and direct sequencing. RESULTS: Preoperative corneal thickness was normal (539+/-23.13 microm, mean+/-SD), and the photoablation depth was 88.94+/-18.64 microm (mean+/-SD). The most severe corneal haze was grade 2.0 on Hanna's scale one year after PRK. In vivo confocal microscopy also showed normal endothelial cell density and morphology. Aside from an intronic polymorphism in a control, no genetic alterations were found in the lumican and keratocan genes. CONCLUSIONS: There was no evidence that endothelial dysfunction and germline mutation of lumican and keratocan genes participate in the etiology of subepithelial corneal haze. Our findings suggest that the mechanisms of the development of severe corneal opacity are different in humans after PRK compared to the lumican deficient knockout mouse model.

Adult↗
PubMed2006

Interactions between conserved residues in transmembrane helices 2 and 7 during angiotensin AT1 receptor activation.

Site-directed mutagenesis studies and independent molecular modeling studies were combined to investigate the network of inter-residue interactions within the transmembrane region of the angiotensin AT(1a) receptor. Site-directed mutagenesis was focused on residues Tyr292, Asn294, Asn295, and Asn298 in transmembrane helix 7, and the conserved Asp74 in helix 2 and other polar residues. Functional interactions between pairs of residues were evaluated by determining the effects of single and double-reciprocal mutations on agonist-induced AT(1a) receptor activation. Replacement of Tyr292 by aspartate in helix 7 abolished radioligand binding to both Y292D and D74Y/Y292D mutant receptors. Reciprocal mutations of Asp74/Asn294, Ser115/Asn294, Ser252/Asn294, and Asn298/Sen115 caused additive impairment of function, suggesting that these pairs of residues make independent contributions to AT(1a) receptor activation. In contrast, mutations of the Asp74/Tyr298 pair revealed that the D74N/N298D reciprocal mutation substantially increased the impaired inositol phosphate responses of the D74N and N298D receptors. Extensive molecular modeling yielded 3D models of the TM region of the AT(1) receptor and the mutants as well as of their complexes with angiotensin II, which were used to rationalize the possible reasons of impairing of function of some mutants. These data indicate that Asp74 and Asn298 are not optimally positioned for direct strong interaction in the resting conformation of the AT(1a) receptor. Balance of interactions between residues in helix 2 (as D74) and helix 7 (as N294, N295 and N298) in the AT(1) receptors, however, has a crucial role both in determining their functional activity and levels of their expression.

Amino Acids↗
PubMed2005

Differential beta-arrestin binding of AT1 and AT2 angiotensin receptors.

Agonist stimulation of G protein-coupled receptors causes receptor activation, phosphorylation, beta-arrestin binding and receptor internalization. Angiotensin II (AngII) causes rapid internalization of the AT1 receptors, whereas AngII-bound AT2 receptors do not internalize. Although the activation of the rat AT1A receptor with AngII causes translocation of beta-arrestin2 to the receptor, no association of this molecule with the AT2 receptor can be detected after AngII treatment with confocal microscopy or bioluminescence resonance energy transfer. These data demonstrate that the two subtypes of angiotensin receptors have different mechanisms of regulation.

Angiotensin II↗
PubMed2005

[Clinical symptoms, diagnosis and treatment of multiple endocrine neoplasia type 1. Results of genetic screening in Hungarian patients].

Multiple endocrine neoplasia type 1 syndrome is an autosomal dominant disorder characterized by endocrinopathies involving the parathyroid glands, anterior pituitary gland, and pancreas. Also, it may be associated with foregut carcinoid, adrenocortical tumors and non-endocrine tumors. After reviewing the prevalence, genetic background, clinical symptoms, diagnosis and treatment of the disorder, the authors present their genetic screening method used for the detection of mutations of the MEN1 gene (prescreening of polymerase chain reaction amplified exons using temporal temperature gradient gel electrophoresis followed by direct DNA sequencing). Using this method, the authors identified disease-causing MEN1 gene mutations in 9 probands (small deletions in 2 cases, insertion in 2 cases, nonsense mutations in 2 cases and missense mutations in 3 cases). Of the 9 mutations, 4 proved to be novel mutation not reported in the literature. Family screening indicated de novo mutations in 2 probands. In addition to mutations, several sequence polymorphisms were also detected. The authors conclude that one of the major advantages of genetic screening in families with MEN1 syndrome was the identification of family members carrying the mutation who should be regularly screened for disease manifestations and those not carrying the mutation in whom clinical screening is unnecessary. Also, genetic screening may be useful in cases when MEN1 syndrome is suspected, but the clinical manifestations do not fully establish the diagnosis of MEN1 syndrome.

Adrenal Cortex Neoplasms↗
PubMed2005

Selective cellular effects of overexpressed pleckstrin-homology domains that recognize PtdIns(3,4,5)P3 suggest their interaction with protein binding partners.

Several pleckstrin-homology (PH) domains with the ability to bind phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3, PIP3] were expressed as green fluorescent protein (GFP) fusion proteins to determine their effects on various cellular responses known to be activated by PIP3. These proteins comprised the PH domains of Akt, ARNO, Btk or GRP1, and were found to show growth-factor-stimulated and wortmannin-sensitive translocation from the cytosol to the plasma membrane in several cell types, indicating their ability to recognize PIP3. Remarkably, although overexpressed Akt-PH-GFP and Btk-PH-GFP were quite potent in antagonizing the PIP3-mediated activation of the Akt protein kinase, such inhibition was not observed with the other PH domains. By contrast, expression of the PH domains of GRP1 and ARNO, but not of Akt or Btk, inhibited the attachment and spreading of freshly seeded cells to culture dishes. Activation of PLCgamma by epidermal growth factor (EGF) was attenuated by the PH domains of GRP1, ARNO and Akt, but was significantly enhanced by the Btk PH domain. By following the kinetics of expression of the various GFP-fused PH domains for several days, only the PH domain of Akt showed a lipid-binding-dependent self-elimination, consistent with its interference with the anti-apoptotic Akt signaling pathway. Mutations of selective residues that do not directly participate in PIP3 binding in the GRP1-PH and Akt-PH domain were able to reduce the dominant-negative effects of these constructs yet retain their lipid binding. These data suggest that interaction with and sequestration of PIP3 may not be the sole mechanism by which PH domains interfere with cellular responses and that their interaction with other membrane components, most probably with proteins, allows a more specific participation in the regulation of specific signaling pathways.

Androstadienes↗
PubMed2005

Pleiotropic AT1 receptor signaling pathways mediating physiological and pathogenic actions of angiotensin II.

Angiotensin II (Ang II) activates a wide spectrum of signaling responses via the AT1 receptor (AT1R) that mediate its physiological control of blood pressure, thirst, and sodium balance and its diverse pathological actions in cardiovascular, renal, and other cell types. Ang II-induced AT1R activation via Gq/11 stimulates phospholipases A2, C, and D, and activates inositol trisphosphate/Ca2+ signaling, protein kinase C isoforms, and MAPKs, as well as several tyrosine kinases (Pyk2, Src, Tyk2, FAK), scaffold proteins (G protein-coupled receptor kinase-interacting protein 1, p130Cas, paxillin, vinculin), receptor tyrosine kinases, and the nuclear factor-kappaB pathway. The AT1R also signals via Gi/o and G11/12 and stimulates G protein-independent signaling pathways, such as beta-arrestin-mediated MAPK activation and the Jak/STAT. Alterations in homo- or heterodimerization of the AT1R may also contribute to its pathophysiological roles. Many of the deleterious actions of AT1R activation are initiated by locally generated, rather than circulating, Ang II and are concomitant with the harmful effects of aldosterone in the cardiovascular system. AT1R-mediated overproduction of reactive oxygen species has potent growth-promoting, proinflammatory, and profibrotic actions by exerting positive feedback effects that amplify its signaling in cardiovascular cells, leukocytes, and monocytes. In addition to its roles in cardiovascular and renal disease, agonist-induced activation of the AT1R also participates in the development of metabolic diseases and promotes tumor progression and metastasis through its growth-promoting and proangiogenic activities. The recognition of Ang II's pathogenic actions is leading to novel clinical applications of angiotensin-converting enzyme inhibitors and AT1R antagonists, in addition to their established therapeutic actions in essential hypertension.

Angiotensin II↗
PubMed2005

Targeted expression of the inositol 1,4,5-triphosphate receptor (IP3R) ligand-binding domain releases Ca2+ via endogenous IP3R channels.

Virtually all functions of a cell are influenced by cytoplasmic [Ca(2+)] increases. Inositol 1,4,5-trisphosphate receptor (IP(3)R) channels, located in the endoplasmic reticulum (ER), release Ca(2+) in response to binding of the second messenger, IP(3).IP(3)Rs thus are part of the information chain interpreting external signals and transforming them into cytoplasmic Ca(2+) transients. IP(3)Rs function as tetramers, each unit comprising an N-terminal ligand-binding domain (LBD) and a C-terminal channel domain linked by a long regulatory region. It is not yet understood how the binding of IP(3) to the LBD regulates the gating properties of the channel. Here, we use the expression of IP(3) binding protein domains tethered to the surface of the endoplasmic reticulum (ER) to show that the all-helical domain of the IP(3)R LBD is capable of depleting the ER Ca(2+) pools by opening the endogenous IP(3)Rs, even without IP(3) binding. This effect requires the domain to be within 50 A of the ER membrane and is impaired by the presence of the N-terminal inhibitory segment on the LBD. These findings raise the possibility that the helical domain of the LBD functions as an effector module possibly interacting with the channel domain, thereby being part of the gating mechanisms by which the IP(3)-induced conformational change within the LBD regulates Ca(2+) release.

Animals↗
PubMed2005

Ligand binding and functional properties of human angiotensin AT1 receptors in transiently and stably expressed CHO-K1 cells.

Chinese Hamster Ovary Cells (CHO-K1) were transiently and stably transfected to express the human angiotensin AT(1) receptor. Cell surface receptor expression was maximal 2 days after transient transfection. Their pharmacological and signalling properties differed from stably expressed receptors. Receptor reserve was significant in the transient cells but not in stable cells, explaining the higher potency of angiotensin II and the lower degree of insurmountable inhibition by candesartan in the transient cells. [Sar(1)Ile(8)]angiotensin II (sarile) is a potent angiotensin AT(1) receptor antagonist for the stable cells but is a partial agonist, producing 19% of the maximal response by angiotensin II, in transient cells. Internalization of [(3)H]angiotensin II and [(125)I]sarile (i.e., acid-resistant binding) was more pronounced in stable cells. CHO-K1 cells were also transiently transfected with the enhanced green fluorescence-AT(1) receptor gene. Confocal microscopy revealed rapid internalization induced by angiotensin II and sarile but not by candesartan. The above disparities may result from differences in receptor maturation and/or cellular surrounding.

1-Sarcosine-8-Isoleucine Angiotensin II↗
PubMed2004

Unusual presentation of multiple endocrine neoplasia type 1 in a young woman with a novel mutation of the MEN1 gene.

We report an unusual presentation of multiple endocrine neoplasia type 1 (MEN 1) in a young woman who was subsequently proven to have a novel mutation of the MEN1 gene. The young patient, aged 25 years, was investigated for abdominal discomfort and left upper abdominal pain. Her family history was unremarkable, except an unknown disorder of her father causing early death. Abdominal ultrasonography (USG) and computed tomography revealed a giant pancreatic tumor measuring 10 cm in diameter. The diagnosis of a clinically nonfunctioning pancreatic neuroendocrine tumor was established by clinical and other studies, including USG-guided aspiration biopsy and octreotide scintigraphy, and the patient underwent a distal pancreatectomy. Histology proved a well-differentiated multinodular neuroendocrine tumor of the pancreas. During surgery, a subcutaneous lipoma was also removed from the abdominal wall. Two years later, the patient developed primary hyperparathyroidism, and two enlarged parathyroid glands were surgically removed. Magnetic resonance imaging of the pituitary gland was normal. Screening for MEN1 gene mutation by temperature gradient gel electrophoresis revealed heterozygosities in exons 3, 8, and 9, while direct sequencing indicated a novel germline mutation (C354X) resulting in a stop codon in exon 8 and polymorphisms in exon 3 (R171Q) and exon 9 (D418D and L432L). Genetic screening revealed no mutation in living family members. Our unusual case suggests that a multinodular pancreatic neuroendocrine tumor in a young patient may justify screening for MEN 1 syndrome, even in the absence of other endocrinopathy or family history.

Adult↗
PubMed2004

Differential pathways of angiotensin II-induced extracellularly regulated kinase 1/2 phosphorylation in specific cell types: role of heparin-binding epidermal growth factor.

Stimulation of the angiotensin II (Ang II) type 1 receptor (AT1-R) causes phosphorylation of extracellularly regulated kinases 1 and 2 (ERK1/2) via epidermal growth factor receptor (EGF-R) transactivation-dependent or -independent pathways in Ang II target cells. Here we examined the mechanisms involved in agonist-induced EGF-R transactivation and subsequent ERK1/2 phosphorylation in clone 9 (C9) hepatocytes, which express endogenous AT1-R, and COS-7 and human embryonic kidney (HEK) 293 cells transfected with the AT1-R. Ang II-induced ERK1/2 activation was attenuated by inhibition of Src kinase and of matrix metalloproteinases (MMPs) in C9 and COS-7 cells, but not in HEK 293 cells. Agonist-mediated MMP activation in C9 cells led to shedding of heparin-binding EGF (HB-EGF) and stimulation of ERK1/2 phosphorylation. Blockade of HB-EGF action by neutralizing antibody or its selective inhibitor, CRM197, attenuated ERK1/2 activation by Ang II. Consistent with its agonist action, HB-EGF stimulation of these cells caused marked phosphorylation of the EGF-R and its adapter molecule, Shc, as well as ERK1/2 and its dependent protein, p90 ribosomal S6 kinase, in a manner similar to that elicited by Ang II or EGF. Although the Tyr319 residue of the AT1-R has been proposed to be an essential regulator of EGF-R transactivation, stimulation of wild-type and mutant (Y319F) AT1-R expressed in COS-7 cells caused EGF-R transactivation and subsequent ERK1/2 phosphorylation through release of HB-EGF in a Src-dependent manner. In contrast, the noninvolvement of MMPs in HEK 293 cells, which may reflect the absence of Src activation by Ang II, was associated with lack of transactivation of the EGF-R. These data demonstrate that the individual actions of Ang II on EGF-R transactivation in specific cell types are related to differential involvement of MMP-dependent HB-EGF release.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Structural determinants of agonist-induced signaling and regulation of the angiotensin AT1 receptor.

Angiotensin II (Ang II) regulates aldosterone secretion by stimulating inositol phosphate production and Ca(2+) signaling in adrenal glomerulosa cells via the G(q)-coupled AT(1) receptor, which is rapidly internalized upon agonist binding. Ang II also binds to the heptahelical AT(2) receptor, which neither activates inositol phosphate signaling nor undergoes receptor internalization. The differential behaviors of the AT(1) and AT(2) receptors were analyzed in chimeric angiotensin receptors created by swapping the second (IL2), the third (IL3) intracellular loops and/or the cytoplasmic tail (CT) between these receptors. When transiently expressed in COS-7 cells, the chimeric receptors showed only minor alterations in their ligand binding properties. Measurements of the internalization kinetics and inositol phosphate responses of chimeric AT(1A) receptors indicated that the CT is required for normal receptor internalization, and IL2 is a determinant of G protein activation. In addition, the amino-terminal portion of IL3 is required for both receptor functions. However, only substitution of IL2 impaired Ang II-induced ERK activation, suggesting that alternative mechanisms are responsible for ERK activation in signaling-deficient mutant AT(1) receptors. Substitution of IL2, IL3, or CT of the AT(1A) receptor into the AT(2) receptor sequence did not endow the latter with the ability to internalize or to mediate inositol phosphate signaling responses. These data suggest that the lack of receptor internalization and inositol phosphate signal generation by the AT(2) receptor is a consequence of its different activation mechanism, rather than the inability of its cytoplasmic domains to couple to intracellular effectors.

Amino Acid Sequence↗
PubMed2004

Intracellular trafficking of hormone receptors.

Agonist binding stimulates endocytosis of hormone receptors via vesicular uptake mechanisms. Interactions of the intracellular domains of receptors with specific targeting proteins are crucial for sorting of internalized receptor in endosomes. Some receptors are targeted for very rapid (e.g. beta2-adrenergic receptor) or slower (e.g. AT1 angiotensin receptor) recycling pathways, whereas others are targeted to lysosomes for degradation (e.g. EGF receptor or PAR1 protease-activated receptor). This review discusses the mechanisms involved in these processes, which regulate surface receptor expression and set the stage for intracellular signaling of G protein-coupled and growth factor receptors.

Animals↗
PubMed2004

The role of the AT1 angiotensin receptor in cardiac hypertrophy: angiotensin II receptor or stretch sensor?

Activation of the AT(1) angiotensin receptor is a clinically important maladaptive response during cardiac hypertrophy. Autocrine and paracrine effects of locally generated angiotensin II, are believed to be the main mediators of these responses. However, a recent report has suggested that mechanical stress can activate AT(1) receptors independently of angiotensin II generation. This finding, as well as recent studies on intracrine effects and the pharmacological consequences of receptor hetero-oligomerization, suggest that unexpected mechanisms could contribute to the role of the renin-angiotensin system during cardiac hypertrophy.

Animals↗
PubMed2004

Control of aldosterone secretion: a model for convergence in cellular signaling pathways.

Aldosterone secretion by glomerulosa cells is stimulated by angiotensin II (ANG II), extracellular K(+), corticotrophin, and several paracrine factors. Electrophysiological, fluorimetric, and molecular biological techniques have significantly clarified the molecular action of these stimuli. The steroidogenic effect of corticotrophin is mediated by adenylyl cyclase, whereas potassium activates voltage-operated Ca(2+) channels. ANG II, bound to AT(1) receptors, acts through the inositol 1,4,5-trisphosphate (IP(3))-Ca(2+)/calmodulin system. All three types of IP(3) receptors are coexpressed, rendering a complex control of Ca(2+) release possible. Ca(2+) release is followed by both capacitative and voltage-activated Ca(2+) influx. ANG II inhibits the background K(+) channel TASK and Na(+)-K(+)-ATPase, and the ensuing depolarization activates T-type (Ca(v)3.2) Ca(2+) channels. Activation of protein kinase C by diacylglycerol (DAG) inhibits aldosterone production, whereas the arachidonate released from DAG in ANG II-stimulated cells is converted by lipoxygenase to 12-hydroxyeicosatetraenoic acid, which may also induce Ca(2+) signaling. Feedback effects and cross-talk of signal-transducing pathways sensitize glomerulosa cells to low-intensity stimuli, such as physiological elevations of [K(+)] (< or =1 mM), ANG II, and ACTH. Ca(2+) signaling is also modified by cell swelling, as well as receptor desensitization, resensitization, and downregulation. Long-term regulation of glomerulosa cells involves cell growth and proliferation and induction of steroidogenic enzymes. Ca(2+), receptor, and nonreceptor tyrosine kinases and mitogen-activated kinases participate in these processes. Ca(2+)- and cAMP-dependent phosphorylation induce the transfer of the steroid precursor cholesterol from the cytoplasm to the inner mitochondrial membrane. Ca(2+) signaling, transferred into the mitochondria, stimulates the reduction of pyridine nucleotides.

Aldosterone↗
PubMed2003

Independent beta-arrestin 2 and G protein-mediated pathways for angiotensin II activation of extracellular signal-regulated kinases 1 and 2.

Stimulation of a mutant angiotensin type 1A receptor (DRY/AAY) with angiotensin II (Ang II) or of a wild-type receptor with an Ang II analog ([sarcosine1,Ile4,Ile8]Ang II) fails to activate classical heterotrimeric G protein signaling but does lead to recruitment of beta-arrestin 2-GFP and activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) (maximum stimulation approximately 50% of wild type). This G protein-independent activation of mitogen-activated protein kinase is abolished by depletion of cellular beta-arrestin 2 but is unaffected by the PKC inhibitor Ro-31-8425. In parallel, stimulation of the wild-type angiotensin type 1A receptor with Ang II robustly stimulates ERK1/2 activation with approximately 60% of the response blocked by the PKC inhibitor (G protein dependent) and the rest of the response blocked by depletion of cellular beta-arrestin 2 by small interfering RNA (beta-arrestin dependent). These findings imply the existence of independent G protein- and beta-arrestin 2-mediated pathways leading to ERK1/2 activation and the existence of distinct "active" conformations of a seven-membrane-spanning receptor coupled to each.

Amino Acid Sequence↗