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L Hunyady

Publications and source records attributed to L Hunyady.

At least 37 records · Page 2Linked to original sources
PubMed1997

[AT1 angiotensin receptor inhibition as a new therapeutic possibility].

The octapeptide hormone, angiotensin II, binds to two major subtypes of cell surface receptors: the AT1 and the AT2 angiotensin receptors. The important physiological and pathophysiological effects of angiotensin II on cardiovascular regulation and salt-water balance are mediated by the AT1 receptor subtype. As a consequence of the outstanding clinical success of angiotensin-converting enzyme inhibitors, the appearance of AT1 receptor inhibitors in the therapy of hypertension and other cardiovascular diseases was preceded with great expectations. The available experimental and clinical data indicate that the first AT1 receptor inhibitor, losartan, has the same therapeutic potential as angiotensin-converting enzyme inhibitors, but it does not evoke the angiotensin-independent side-effects of ACE inhibitors, such as dry cough or angioedema. The physiological importance and the biochemical, molecular biological and pharmacological properties of AT1 and AT2 receptors are reviewed in this paper, and a summary of the available clinical data is presented.

Angiotensin I↗
PubMed1996

Dependence of agonist activation on a conserved apolar residue in the third intracellular loop of the AT1 angiotensin receptor.

The coupling of agonist-activated seven transmembrane domain receptors to G proteins is known to involve the amino-terminal region of their third cytoplasmic loop. Analysis of the amino acids in this region of the rat type in angiotensin (AT1a) receptor identified Leu-222 as an essential residue in receptor activation by the physiological agonist, angiotensin II (Ang II). Nonpolar replacements for Leu-222 yielded functionally intact AT1 receptors, while polar or charged residues caused progressive impairment of Ang II-induced inositol phosphate generation. The decrease in agonist-induced signal generation was associated with a parallel reduction of receptor internalization, and was most pronounced for the Lys-222 mutant receptor. Although this mutant showed normal binding of the peptide antagonist, [Sar1,Ile6]Ang II, its affinity for Ang II was markedly reduced, consistent with its inability to adopt the high-affinity conformation. A search revealed that many Gq-coupled receptors contain an apolar amino acid (frequently leucine) in the position corresponding to Leu-222 of the AT1 receptor. These findings suggest that such a conserved apolar residue in the third intracellular loop is a crucial element in the agonist-induced activation of the AT1 and possibly many other G protein-coupled receptors.

Amino Acid Sequence↗
PubMed1996

The ligand binding site of the angiotensin AT1 receptor.

The angiotensin AT1 receptor mediates numerous physiological actions of the octapeptide hormone, angiotensin II, in its cardiovascular and other target tissues. The binding of angiotensin II to the AT1 receptor is dependent on both intramembrane and extracellular regions of the receptor molecule. Non-peptide antagonists that block angiotensin binding and action interact exclusively with residues located within the intramembrane binding pocket of the AT1 receptor. However, peptide ligands also interact with extracellular residues to form additional bonds that stabilize their binding to the receptor. Here, László Hunyady, Tamás Balla and Kevin Catt describe how these and other studies have shown that interaction of ligands with residues in the intramembrane binding pocket is the conserved mechanism required for agonist activation of G protein-coupled receptors.

Angiotensin I↗
PubMed1995

A conserved NPLFY sequence contributes to agonist binding and signal transduction but is not an internalization signal for the type 1 angiotensin II receptor.

A conserved NPX2-3Y sequence that is located in the seventh transmembrane helix of many G protein-coupled receptors has been predicted to participate in receptor signaling and endocytosis. The role of this sequence (NPLFY) in angiotensin II receptor function was studied in mutant and wild-type rat type 1a angiotensin II receptors transiently expressed in COS-7 cells. The ability of the receptor to interact with G proteins and to stimulate inositol phosphate responses was markedly impaired by alanine replacement of Asn298 and was reduced by replacement of Pro299 or Tyr302. The F301A mutant receptor exhibited normal G protein coupling and inositol phosphate responses, and the binding of the peptide antagonist, [Sar1,Ile8]angiotensin II, was only slightly affected. However, its affinity for angiotensin II and the nonpeptide antagonist losartan was reduced by an order of a magnitude, suggesting that angiotensin II and losartan share an intramembrane binding site, possibly through their aromatic moieties. None of the agonist-occupied mutant receptors, including Y302A and triple alanine replacements of Phe301, Tyr302, and Phe304, showed substantial changes in their internalization kinetics. These findings demonstrate that the NPLFY sequence of the type 1a angiotensin II receptor is not an important determinant of agonist-induced internalization. However, the Phe301 residue contributes significantly to agonist binding, and Asn298 is required for normal receptor activation and signal transduction.

1-Sarcosine-8-Isoleucine Angiotensin II↗
PubMed1995

Critical role of a conserved intramembrane tyrosine residue in angiotensin II receptor activation.

The rat type 1a (AT1a) angiotensin II (Ang II) receptor contains a highly conserved tyrosine residue in the fifth transmembrane region that is present in most G protein-coupled receptors. The role of this amino acid in AT1 receptor activation was analyzed in a mutant receptor (Y215F) created by replacing Tyr215 with phenylalanine. The mutant receptor was highly expressed in transfected COS-7 cells, and its binding affinity for the peptide antagonist [Sar1,Ile8]Ang II was similar to that of the wild type receptor. Although the structural integrity of the peptide ligand binding domain was preserved in the Y215F mutant receptor, its affinity for the native agonist, Ang II, was significantly reduced. Also, whereas guanosine 5'-3-O-(thio)triphosphate markedly reduced Ang II binding to the wild type receptor, it had little effect on agonist binding to the mutant receptor. Agonist-induced internalization of the mutant receptor was also impaired, and its ability to mediate inositol phosphate responses to Ang II stimulation was abolished. The concomitant decreases in receptor internalization and G protein-mediated signaling of the Y215F mutant receptor indicate that Tyr215 has a critical role in AT1 receptor activation. In view of its conservation among members of the seven transmembrane domain receptor superfamily, this residue is likely to be of general importance in signal transduction from G protein-coupled receptors.

Amino Acid Sequence↗
PubMed1994

Identification of a cytoplasmic Ser-Thr-Leu motif that determines agonist-induced internalization of the AT1 angiotensin receptor.

The type 1 angiotensin II (AT1) receptor undergoes rapid endocytosis and down-regulation after agonist binding. In studies on the structural determinants of agonist-induced endocytosis, serial deletions in the cytoplasmic tail of the rat AT1a receptor showed that the carboxyl-terminal 22 amino acids are not necessary for its internalization. However, internalization was markedly impaired by the removal of one additional amino acid (Leu337) and was reduced by 95% after removal of Ser335 and Thr336. Single alanine replacements of amino acids in this region showed that individual substitutions of Thr332, Ser335, Thr336, Leu337, and Ser338 caused moderate but significant impairment of the internalization rate. Replacement of both Ser335 and Thr336 with alanine residues further impaired the internalization rate, and triple alanine replacement of the Ser-Thr-Leu motif reduced internalization to almost the same extent as the corresponding tail deletion mutant. The Ser-Thr-Leu motif is highly conserved in mammalian AT1 receptors but is not present in the noninternalizing type 2 angiotensin II receptor. These data demonstrate that a serine/threonine-rich region including Leu337 in the cytoplasmic tail of the AT1 receptor is a major requirement for endocytosis of the hormone-receptor complex and support the concept that similar motifs in other G protein-coupled receptors are determinants of their agonist-induced internalization.

Amino Acid Sequence↗
PubMed1994

Evidence for participation of calcineurin in potentiation of agonist-stimulated cyclic AMP formation by the calcium-mobilizing hormone, angiotensin II.

Angiotensin II (AII) receptors are known to interact with two distinct guanine nucleotide binding proteins, Gq/11 and Gi, in rat adrenal glomerulosa cells to activate phospholipase C and to inhibit adenylate cyclase, respectively. However, in cultured bovine glomerulosa cells AII potentiates rather than inhibits the stimulatory effect of adrenocorticotropin (ACTH) on cAMP levels. This effect of AII was partially mimicked by phorbol 12-myristate 13-acetate (PMA) and was partially inhibited by staurosporine or depletion of protein kinase C but was unaffected by pertussis toxin treatment. No potentiation was detectable in disrupted cells or in membrane preparations. In intact glomerulosa cells, treatment with cyclosporin A or FK506 completely inhibited AII- or PMA-induced potentiation of cAMP production without affecting the response to ACTH. In COS-7 cells transfected with the rat AT1 receptor, AII caused 2-3-fold enhancement of the ACTH-induced cAMP response, an effect that was partially reproduced by PMA. These potentiating actions of AII and PMA were prevented by preincubation with cyclosporin A or FK506, and the latter effect was abolished by rapamycin. These results implicate the Ca2+- and calmodulin-dependent protein phosphatase, calcineurin, in AII-induced enhancement of adenylate cyclase activity in both adrenal glomerulosa and transfected COS-7 cells. The finding that AII enhances ACTH-stimulated production of cAMP by a second messenger-mediated mechanism that involves the participation of calcineurin reveals an additional mode of cross-talk between pathways activated by Ca(2+)-mobilizing and cAMP-generating receptors.

Adrenocorticotropic Hormone↗
PubMed1994

Independence of type I angiotensin II receptor endocytosis from G protein coupling and signal transduction.

The relationship between angiotensin II-induced activation of G proteins and receptor internalization was analyzed by transiently expressing mutant and wild type cDNAs for the rat AT1a receptor in COS-7 cells. Pertussis toxin-sensitive G proteins did not appear to play a role in endocytosis since the receptor showed normal internalization kinetics in pertussis toxin-treated cells. Three deletion mutants of the third cytoplasmic loop revealed that the N-terminal part of this region is important for both receptor endocytosis and intracellular signaling. Three point mutations of Asp74, which has been implicated in signal transduction by the AT1a receptor, caused impaired G protein coupling and inositol phosphate responses. However, each of these mutants (D74N, D74H, and D74Y) showed markedly different internalization kinetics. The D74Y mutant showed the greatest impairment of internalization but retained the highest degree of inositol phosphate stimulation. In contrast, the D74N mutant, which showed the most impaired G protein coupling and inositol phosphate responses, had similar internalization kinetics to the wild type receptor. The combined mutant receptor containing the D74N substitution and deletion of residues 221-226 from the third cytoplasmic loop showed no G protein coupling or inositol phosphate response but was internalized about 60% as rapidly as the wild type receptor. These data demonstrate that endocytosis of the AT1 receptor is independent of agonist-activated signal transduction and indicate that receptor internalization and activation of phospholipase C have different structural requirements.

Angiotensin II↗
PubMed1994

Plasmalemmal dihydropyridine receptors modify the function of subplasmalemmal inositol 1,4,5-trisphosphate receptors: a hypothesis.

Experimental observations on rat glomerulosa cells inspired a model which postulates that plasmalemmal dihydropyridine receptors are in juxtaposition and interaction with inositol 1,4,5-trisphosphate receptors in subplasmalemmal calciosomes. Activation of dihydropyridine receptors promotes the Ca2+ releasing effect of inositol 1,4,5-trisphosphate. The most important observations compatible with the model are the following: (1) angiotensin II does not influence Ca2+ influx during the peak phase of Ca2+ signal; (2) dihydropyridine drugs modify the initial peak of the Ca2+ signal induced by angiotensin II; (3) inhibitors of the dihydropyridine receptor reduce the initial Ca2+ signal also in the presence of 5 mM Ni2+, an inhibitor of voltage dependent Ca2+ influx; and (4) changes in extracellular K+ concentration within the physiological range also modify the cytoplasmic Ca2+ response to angiotensin II.

Angiotensin II↗
PubMed1994

Dihydropyridine-sensitive initial component of the ANG II-induced Ca2+ response in rat adrenal glomerulosa cells.

The Ca2+ signal induced by an increase in extracellular K+ concentration from 3.6 to 5.6 mM or angiotensin II (ANG II) was inhibited by the dihydropyridine (DHP) Ca2+ channel blocker, nifedipine, and enhanced by the DHP Ca2+ channel agonist, BAY K 8644. The DHP sensitivity of the ANG II-induced Ca2+ response was already detectable during the peak phase, suggesting that the DHP receptor plays an important role during the initial phase of ANG II stimulation. K+ and ANG II stimulated a nifedipine-sensitive Mn2+ influx pathway, further promoting the role of a DHP receptor in their mechanism of action. Fluorescent membrane potential measurements showed that, in contrast to the rapid depolarization induced by K+, the ANG II-induced depolarization had a lag time of > 30 s. The slow kinetics of depolarization compared with the immediate effect of ANG II on Mn2+ influx and the DHP sensitivity of the initial Ca2+ peak indicates that ANG II initiates the activation of the DHP-sensitive Ca2+ channel by a mechanism other than depolarization.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1994

Capacitative Ca2+ influx in adrenal glomerulosa cells: possible role in angiotensin II response.

We examined the effect of the depletion of intracellular Ca2+ stores on Ca2+ influx in rat glomerulosa cells. Depletion of intracellular Ca2+ stores was achieved by inhibiting sarco/endoplasmic reticulumtype Ca(2+)-ATPase with thapsigargin or 2,5,di-(t-butyl)-1,4-benzohydroquinone (t-BHQ). Both inhibitors induced a sustained rise in cytoplasmic Ca2+ concentration. The initial rise was observed also in Ca(2+)-free medium, while the sustained phase disappeared, indicating that the latter requires Ca2+ influx. In Ca(2+)-free medium, the readdition of Ca2+ induced a steeper and higher rise in intracellular Ca2+ concentration in thapsigargin-treated cells than in controls, supporting the role of Ca2+ influx. In normal medium, the addition of Cd2+ (80 microM) evoked an immediate inhibition of the sustained phase of thapsigargin response. The response to thapsigargin was insensitive to nifedipine. Thapsigargin failed to enhance Mn2+ quenching of fura 2. Our results provide evidence for the existence of capacitative Ca2+ influx in rat glomerulosa cells and indicate that dihydropyridine-sensitive Ca2+ channels do not participate in capacitative Ca2+ entry. High concentrations of thapsigargin and t-BHQ, similar to the reported effects of angiotensin II and vasopressin, inhibited K(+)-induced Ca2+ signals. These effects appear, however, to be independent of the depletion of internal Ca2+ stores.

Angiotensin II↗
PubMed1994

Sequential activation of phospholipase-C and -D in agonist-stimulated gonadotrophs.

The contributions of phospholipase-C and -D to diacylglycerol (DG) formation during agonist-induced cell signaling were investigated in rat pituitary cells and alpha T3-1 gonadotrophs. In both cell types, GnRH caused a biphasic increase in DG formation, with an initial spike within 60 sec, followed by a larger and sustained rise to reach a second peak after 15 min of stimulation. Both phases of DG production were temporally correlated with inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] formation, consistent with the dependence of DG formation on phospholipase-C-mediated phosphoinositide hydrolysis. However, the ability of GnRH to stimulate phosphatidylethanol (PEt) in the presence of ethanol suggested that phospholipase-D may also participate in DG formation. Two inhibitors of phospholipase-C-dependent phosphoinositide hydrolysis, U73122 and neomycin sulfate, reduced the PEt as well as the Ins(1,4,5)P3 response to GnRH, indicating that phospholipase-D is activated during phospholipase-C-dependent signaling in pituitary gonadotrophs. The production of both DG and PEt was increased by treatment with the active phorbol ester phorbol 12-myristate 13-acetate (PMA), but not with inactive 4 alpha-phorbol 13-didecanoate, indicating that stimulation of protein kinase-C leads to activation of phospholipase-D. In accord with this, GnRH- and PMA-induced elevations of DG and PEt production were attenuated or abolished in protein kinase-C-depleted cells. In contrast, short and long term stimulation with PMA had no effect on basal inositol phosphate production. Also, GnRH-induced inositol phosphate production was not affected by protein kinase-C depletion. Finally, U73122 and neomycin sulfate did not inhibit PMA-induced PEt formation. These data indicate that GnRH activates a dual phospholipase pathway in a sequential and synchronized manner; phospholipase-C initiates the biphasic increase in Ins(1,4,5)P3 and DG formation, and protein kinase-C mediates the integration of phospholipase-D into the signaling response during the sustained phase of agonist stimulation.

Animals↗
PubMed1992

Pyridine nucleotide redox state parallels production of aldosterone in potassium-stimulated adrenal glomerulosa cells.

Extracellular potassium ions (K+) raise the intracellular concentration of free Ca2+ ([Ca2+]i) by gating voltage-dependent Ca2+ channels and stimulate aldosterone production in adrenal glomerulosa cells. The pathway leading from calcium influx to increased steroid synthesis has not been completely elucidated. In the present study we demonstrate that the reduction of pyridine nucleotides known to be required for steroid hydroxylation is enhanced by K+ (4.1-8.4 mM) in single rat glomerulosa cells. The action of K+ was strictly dependent on the presence of extracellular Ca2+. Amytal, a blocker of site I of the mitochondrial respiratory chain, abolished the K+ effect, indicating a mitochondrial origin for the recorded changes. Supraphysiological K+ concentration (18 mM) resulted in a further increase in [Ca2+]i, while steroidogenesis was decreased as measured in cell suspensions. However, a possible explanation for this dichotomy is provided by the finding that the level of reduced pyridine nucleotides also decreased at supraphysiological K+ concentration.

Aldosterone↗
PubMed1992

Angiotensin-II inhibits Na+/K+ pump in rat adrenal glomerulosa cells: possible contribution to stimulation of aldosterone production.

The control of Na+/K+ pump activity was studied in rat adrenal glomerulosa cells. Ninety percent of K+/86Rb accumulation was blocked by ouabain, and the dose-response curve of inhibition by ouabain was monophasic (IC50, approximately 80 microM), suggesting the role of a single type of Na+/K+ pump (alpha-isoenzyme) in 86Rb accumulation by rat glomerulosa cells. The basal activity of the Na+/K+ pump was much higher in glomerulosa cells than in adrenal fasciculata cells or hepatocytes, as judged by the ouabain-sensitive uptake of 86Rb. In contrast to the two other cell types, increasing Na+ influx with the Na+ ionophore monensin failed to significantly affect ouabain-sensitive 86Rb uptake in glomerulosa cells, suggesting that in glomerulosa cells even the resting intracellular Na+ concentration is sufficient for maximal activity of the Na+/K+ pump. Angiotensin-II (AII) inhibited the ouabain-sensitive 86Rb uptake by glomerulosa cells. The effect of AII was abolished by the selective antagonist of the AT1 type of AII receptors (DuP 753), while PD 123177, an AT2 antagonist was ineffective. AT1 receptors of glomerulosa cells coupled to phospholipase-C activation and, thus, to Ca2+ signal. The inhibitory effect of AII was dependent on the extracellular Ca2+ concentration, but an elevation of cytoplasmic Ca2+ by Ca2+ ionophore ionomycin failed to mimic the effect of AII. These data suggest that Ca2+ is required for but does not mediate the inhibitory effect of AII on the Na+/K+pump. Pharmacological activation of protein kinase-C by phorbol ester did not modify 86Rb accumulation by the cells. Ouabain induced a nifedipine-sensitive elevation in the cytoplasmic Ca2+ concentration and exerted a stimulatory effect on aldosterone production, suggesting participation of the inhibition of the Na+/K+ pump in the aldosterone stimulatory action of AII.

Aldosterone↗
PubMed1991

Agonist-induced endocytosis and signal generation in adrenal glomerulosa cells. A potential mechanism for receptor-operated calcium entry.

The relationships between receptor-mediated endocytosis and the generation of intracellular signals were analyzed in angiotensin II (AII)-stimulated adrenal glomerulosa cells. In cells equilibrated with 125I-AII analogs at 4 degrees C, specifically bound agonist but not antagonist AII derivatives were rapidly internalized at 37 degrees C. AII-induced internalization was not influenced by the presence or absence of extracellular Ca2+ but was inhibited by treatment with phenylarsine oxide (PAO) or by arresting coated pit formation with hypotonic shock and potassium depletion. Inhibition of internalization by PAO was prevented by the bifunctional sulfhydryl reagent dithiothreitol but only partially reversed by mercaptoethanol, and readdition of K+ restored internalization in K(+)-depleted cells. Treatment with PAO did not impair the initial AII-induced elevations of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and cytoplasmic calcium [( Ca2+]i) but reduced the sustained phase of the Ins(1,4,5)P3 response by 85% and abolished the second phase of the cytoplasmic Ca2+ response; these responses were restored by concomitant treatment with dithiothreitol. Inhibition of AII-receptor internalization by K+ depletion also caused selective loss of the sustained phase of the AII-induced Ca2+ response. Thus, blockade of AII-receptor internalization has similar effects as extracellular Ca2+ deficiency, which abolishes the sustained but not the early AII-induced increases in Ins(1,4,5)P3 production and [Ca2+]i. The close correlations between AII-induced internalization and the generation of Ins(1,4,5)P3 and [Ca2+]i responses suggest that endocytosis of the agonist-receptor complex is necessary to maintain the production of these intracellular signals. It is also possible that receptor-operated vesicular uptake of extracellular Ca2+ makes a significant contribution to the sustained [Ca2+]i responses of certain agonist-stimulated target cells.

Angiotensin II↗
PubMed1991

Second messengers derived from inositol lipids.

Many hormones, growth factors, and neurotransmitters stimulate their target cells by promoting the hydrolysis of plasma-membrane phosphoinositides to form the two second messengers, diacylglycerol and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. In such cells, ligand-receptor interaction stimulates specific phospholipases that are activated by guanyl nucleotide regulatory G proteins or tyrosine phosphorylation. In many cells, the initial rise in cytoplasmic calcium due to Ins(1,4,5)P3-induced mobilization of calcium from agonist-sensitive stores is followed by a sustained phase of cytoplasmic calcium elevation that maintains the target-cell response, and is dependent on influx of extracellular calcium. Numerous inositol phosphates are formed during metabolism of the calcium-mobilizing messenger, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], to lower and higher phosphorylated derivatives. The cloning of several phospholipase-C isozymes, as well as the Ins(1,4,5)P3-5 kinase and the Ins(1,4,5)P3 receptor, have clarified several aspects of the diversity and complexity of the phosphoinositide-calcium signaling system. In addition to their well-established roles in hormonal activation of cellular responses such as secretion and contraction, phospholipids and their hydrolysis products have been increasingly implicated in the actions of growth factors and oncogenes on cellular growth and proliferation.

Animals↗