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I Lengyel

Publications and source records attributed to I Lengyel.

At least 19 recordsLinked to original sources
PubMed2004

Autonomous activity of CaMKII is only transiently increased following the induction of long-term potentiation in the rat hippocampus.

A major role has been postulated for a maintained increase in the autonomous activity of CaMKII in the expression of long-term potentiation (LTP). However, attempts to inhibit the expression of LTP with CaMKII inhibitors have yielded inconsistent results. Here we compare the changes in CaMKII autonomous activity and phosphorylation at Thr286 of alphaCaMKII in rat hippocampal slices using chemical or tetanic stimulation to produce either LTP or short-term potentiation (STP). Tetanus-induced LTP in area CA1 requires CaMKII activation and Thr286 phosphorylation of alphaCaMKII, but we did not observe an increase in autonomous activity. Next we induced LTP by 10 min exposure to 25 mM tetraethyl-ammonium (TEA) or 5 min exposure to 41 mM potassium (K) after pretreatment with calyculin A. Exposure to K alone produced STP. These protocols allowed us to monitor temporal changes in autonomous activity during and after exposure to the potentiating chemical stimulus. In chemically induced LTP, autonomous activity was maximally increased within 30 s whereas this increase was significantly delayed in STP. However, in both LTP and STP the two-fold increase in autonomous activity measured immediately after stimulation was short-lived, returning to baseline within 2-5 min after re-exposure to normal ACSF. In LTP, but not in STP, the phosphorylation of alphaCaMKII at Thr286 persisted for at least 60 min after stimulation. These results confirm that LTP is associated with a maintained increase in autophosphorylation at Thr286 but indicate that a persistent increase in the autonomous activity of CaMKII is not required for the expression of LTP.

Animals↗
PubMed2004

Enhanced G-protein activation by a mixture of Abeta(25-35), Abeta(1-40/42) and zinc.

Beta-amyloid peptides (Abetas) bind to several G-protein coupled receptor proteins and stimulate GTPase activity in neurons. In this study we determined the effects of Abeta(1-42), Abeta(1-40), Abeta(25-35) and their mixtures on [(35)S]GTP binding in rat brain cortical membranes in the absence and presence of zinc. We found that the Abetas alone induced a concentration-dependent activation of G-proteins (IC50 approximately 10(-6) m), while aggregated Abeta fibrils only affected GTP binding at concentrations above 10(-5) m. Mixing Abeta(25-35) with Abeta(1-42) or Abeta(1-40) induced a several-fold increase in GTP-binding. This potentiation followed a bell shaped curve with a maximum at 50 : 50 ratios. No potentiating effect could be seen by mixing Abeta(1-40) and Abeta(1-42) or highly aggregated Abetas. Zinc had no effect on Abeta(1-40/42) but strongly potentiated the Abeta(25-35) or the mixed peptides-induced GTP-binding. Changes in secondary structure accompanied the mixed peptides or the peptide/zinc complexes induced potentiation, revealing that structural alterations are behind the increased biological action. These concentration dependent potentiating effects of zinc and the peptide mixtures could be physiologically important at brain regions where peptide fragments and/or zinc are present at elevated concentrations.

Amyloid beta-Peptides↗
PubMed2002

Side chain modifications change the binding and agonist properties of endomorphin 2.

Side chain modifications were introduced to endomorphin 2 (E2) to improve its binding properties and biological activity. A number of C-terminal modifications decreased the binding affinity to the mu-opioid receptor and the intrinsic activity in rat brain membranes. The exception was E2-ol, which showed increased binding affinity to MOR and higher potency in stimulating [(35)S]GTPgammaS binding. N-methylation of Phe(3) (MePhe(3)) attenuated the binding affinity and produced a rightward shift of [(35)S]GTPgammaS binding curves. All derivatives had lower intrinsic activity than E2. Some of the modified peptides partially inhibited, while YPF-benzyl-allyl-amide fully inhibited, the E2 or [d-Ala(2),MePhe(4),Gly(5)ol]enkephalin stimulated [(35)S]GTPgammaS binding. Marked differences were found between the results obtained using tritiated E2, tritiated naloxone, and [(35)S]GTPgammaS binding, indicating the possible involvement of multiple binding sites. The data presented demonstrate that the C-terminal amide group has an essential role in the regulation of the binding and the agonist/antagonist properties of E2.

Animals↗
PubMed2001

Influence of degradation on binding properties and biological activity of endomorphin 1.

The recently-isolated endogenous peptide endomorphin 1 has high affinity for the mu opioid receptor and plays an important role in analgesia. Several of its degradation products have been isolated from the central nervous system. Degradation products present structural similarities and may influence the receptor binding properties and biological activity of the parent compound. Therefore, we investigated how degradation of endomorphin 1 might influence ligand binding to the mu opioid receptor, the consequent activation of G proteins and its antinociceptive effect. Both N- and C-terminal truncation of endomorphin 1 resulted in peptides presenting considerably lower opioid receptor binding potency. None of these peptides had an effect on GTP binding, nor was able to produce analgesia, suggesting that degradation destroys the biological activity of endomorphin 1.

Analgesia↗
PubMed2001

Receptor constants for endomorphin-1 and endomorphin-1-ol indicate differences in efficacy and receptor occupancy.

The opioid properties of endomorphin derivatives containing a C-terminal alcoholic(-ol) function were compared to the parent amidated compounds in isolated organs (longitudinal muscle strip of guinea-pig ileum and mouse vas deferens). Similar data were also generated for the mu-opioid receptor selective agonist synthetic peptide (D-Ala2, MePhe4, Gly5-ol)-enkephalin (DAMGO) and its Gly5-NH2 congener (DAMGA). Endomorphin-1-ol (Tyr-Pro-Trp-Phe-ol) had an IC50 of 80.6 nM in mouse vas deferens and 61.2 nM in guinea-pig ileum; the corresponding values for endomorphin-2-ol (Tyr-Pro-Phe-Phe-ol) were 49.6 and 48.2 nM, for DAMGO 59.8 and 29.2 nM, respectively. As it was indicated by the antagonism by naltrexone, the agonist actions were exerted exclusively at mu-opioid receptors in both organs. The -ol derivatives were slightly (2.3-4.3 times) less potent than the parent amides in the bioassays: all peptides had, apparently, full agonist properties in intact preparations. With the aim of revealing potential partial agonist properties among the investigated peptides, we partially inactivated the mu-opioid receptor pool in mouse vas deferens by 5x10(-7) M beta-funaltrexamine. The calculated receptor constants indicated a "high-affinity, low intrinsic efficacy" profile (i.e. a potential partial agonist property) for endomorphin-1, an intermediate character for endomorpin-1-ol and full agonism for DAMGA and DAMGO. Apparently, a higher receptor fraction remained accessible for endomorphin-1 (42.8%) than for the -ol congener (14.0%), DAMGO (20.2%) and DAMGA (14.1%) after partial inactivation.

Analgesics, Opioid↗
PubMed2001

Autonomous activity and autophosphorylation of CAMPK-II in rat hippocampal slices: effects of tissue preparation.

Measurement of the proportion of calcium/calmodulin-stimulated protein kinase II (CaMPK-II) that is autonomously active or phosphorylated on Thr(286) is thought to provide an index of the degree to which CaMPK-II in a tissue has been activated. We have examined how various ways of handling hippocampal tissue can alter these properties. Both autonomous activity and phospho-Thr(286) content was high in freshly dissected hippocampus or freshly cut hippocampal slices. After incubation of hippocampal slices in artificial cerebrospinal fluid for 120 min, both properties of CaMPK-II decreased to a steady state level. Freeze-thaw or cutting the equilibrated slices could rapidly increase both autonomous activity and phospho-Thr(286) immunoreactivity of CaMPK-II. These increases were comparable to changes induced by experimental treatment. Therefore, our results suggest that considerable care needs to be taken over the way in which hippocampal slices are handled.

Animals↗
PubMed2001

Auto-inhibition of Ca(2+)/calmodulin-dependent protein kinase II by its ATP-binding domain.

Ca(2+)/calmodulin dependent protein kinase (CaMPK) II is a key enzyme in many physiological processes. The enzyme is inactive unless Ca(2+)/CaM binds to it. In this inactive form CaMPK-II does not bind ATP suggesting that the ATP-binding domain is involved in an intramolecular interaction. We show here that F12, a 12 amino acid long peptide fragment of the ATP-binding domain (CaMPK-II(23-34), GAFSVVRRCVKV) can inhibit the Ca(2+)/CaM-dependent activity (IC(50) of 3 microM) but has no effect on the Ca(2+)/CaM-independent activity of CaMPK-II. Kinetic analysis exhibited mixed inhibition with respect to autocamtide-2 and ATP. The inhibition by F12 showed specificity towards CaMPK-II, but also inhibited CaMPK-I (IC(50) = 12.5 microM), while CaMPK-IV (IC(50) = 85 microM) was inhibited poorly and cAMP-dependent protein kinase (PKA) was not inhibited. Substitution of phenylalanine at position 25 to alanine (A12), had little effect on the inhibition of different Ca(2+)/CaM-dependent protein kinases, suggesting that phenylalanine 25 does not play a crucial role in the interactions involving F12. Thus the molecular interactions involving the ATP-binding domain appears to play a role in the regulation of nonphosphorylated CaMPK-II activity.

Adenosine Triphosphate↗
PubMed2000

Phosphorylation of a new brain-specific septin, G-septin, by cGMP-dependent protein kinase.

The septins are a family of GTPase enzymes, some of which are required for the cytokinesis stage of cell division and others of which are associated with exocytosis. We purified and cloned the cDNA for a 40-kDa protein from rat brain that is a substrate for type I cGMP-dependent protein kinase (PKG). The amino acid sequences of two tryptic peptides of P40 showed high homology to the septins. Molecular cloning revealed the 358-amino acid P40 to be a new member of the septin family. P40 was named G-septin, as it is phosphorylated in vitro by PKG, but relatively poorly by the related cAMP-dependent protein kinase and not by protein kinase C. Two splice variants of G-septin (alpha and beta) were found with distinct N and C termini, but a common GTPase domain. G-septin lacks the C-terminal coiled-coil domain characteristic of all other mammalian septins and uniquely has two predicted phosphorylation site motifs for type I PKG. Photoaffinity labeling with [alpha-(32)P]GTP confirmed that G-septin is a GTP-binding protein. Northern blotting showed that G-septin mRNA (5.0 kilobases) is highly expressed in brain and undetectable in 12 other tissues, indicating that the G-septins are primarily neuronal proteins. Very low levels of 6.0-, 3.4-, and 2.6-kilobase transcripts were found in testis. Our results reveal a new class of brain-specific septins that may be regulated by PKG in neurons.

Alternative Splicing↗
PubMed2000

Modulation of the phosphorylation and activity of calcium/calmodulin-dependent protein kinase II by zinc.

Calcium/calmodulin-dependent protein kinase II (CaMPK-II) is a key regulatory enzyme in living cells. Modulation of its activity, therefore, could have a major impact on many cellular processes. We found that Zn(2+) has multiple functional effects on CaMPK-II. Zn(2+) generated a Ca(2+)/CaM-independent activity that correlated with the autophosphorylation of Thr(286), inhibited Ca(2+)/CaM binding that correlated with the autophosphorylation of Thr(306), and inhibited CaMPK-II activity at high concentrations that correlated with the autophosphorylation of Ser(279). The relative level of autophosphorylation of these three sites was dependent on the concentration of zinc used. The autophosphorylation of at least these three sites, together with Zn(2+) binding, generated an increased mobility form of CaMPK-II on sodium dodecyl sulfate gels. Overall, autophosphorylation induced by Zn(2+) converts CaMPK-II into a different form than the binding of Ca(2+)/CaM. In certain nerve terminals, where Zn(2+) has been shown to play a neuromodulatory role and is present in high concentrations, Zn(2+) may turn CaMPK-II into a form that would be unable to respond to calcium signals.

Animals↗
PubMed1999

Simultaneous measurement of tyrosine hydroxylase activity and phosphorylation in bovine adrenal chromaffin cells.

A method for simultaneous measurement of tyrosine hydroxylase (TH) activation and phosphorylation in permeabilised and intact bovine adrenal chromaffin cells (BACCs) was established. Permeabilised cells were stimulated with cyclic AMP (1--10 microM) in the presence of [32P]ATP and L-[carboxyl-(14)C]tyrosine. Intact BACCs were preincubated with 32P(i) for 3 h and stimulated with forskolin (1--5 microM) in the presence of L-[carboxyl-(14)C]tyrosine. On stimulation each well was covered with a sealed 'chimney' fitted with a small plastic cup containing 300 microl of 1.0 M NaOH that trapped the 14CO(2) released. TH activity was determined by measuring 14C radioactivity. TH phosphorylation was measured in the same cells by separating the solubilized proteins on SDS PAGE followed by autoradiography and/or HPLC analysis. It was found that H89, a protein kinase A inhibitor, significantly blocked both TH phosphorylation and activation in response to cyclic AMP in permeabilised cells. However, in intact cells, H89 was effective only in respect to forskolin-stimulated TH activity and did not block the forskolin-stimulated TH phosphorylation of Ser-40. The reason(s) for this lack of correlation between TH activation and phosphorylation is presently not understood.

Adrenal Medulla↗
PubMed1999

Phosphorylation of proteins in chick ciliary ganglion under conditions that induce long-lasting changes in synaptic transmission: phosphoprotein targets for nitric oxide action.

Production of nitric oxide and the activation of protein kinases are required for long-term potentiation of synaptic transmission at the giant synapses in chicken ciliary ganglion. In the present study, we investigated the ability of nitric oxide to regulate the phosphorylation of endogenous proteins under conditions that induced long-term potentiation in intact ciliary ganglion and the protein kinases responsible for the phosphorylation of these proteins in lysed ciliary ganglion. Using Calcium Green-1 we showed that the nitric oxide donor sodium nitroprusside did not change the intraterminal Ca2+ dynamics in ciliary ganglion. Two dimensional phosphopeptide analysis of 32Pi-labelled intact ciliary ganglion showed that the sodium nitroprusside (300 microM) increased the phosphorylation of several phosphopeptides (P50a, P50b and P41) derived from proteins at 50,000 and 41,000 mol. wts which we have called nitric oxide-responsive phosphoproteins. A similar stimulation of phosphorylation was achieved by 8-bromo-cyclic AMP (100 microM), which also induced long-term potentiation, but not by phorbol dibutyrate (2 microM) that does not induce long-term potentiation in ciliary ganglion. When subcellular fractions from lysed ciliary ganglion were labelled in vitro by [gamma-32P]ATP in the presence of purified cGMP-dependent, cAMP-dependent or Ca2+-phospholipid-dependent protein kinases, we identified cyclic GMP-dependent protein kinase substrates that gave rise to phosphopeptides co-migrating with P50a, P50b and P41 from 32Pi-labelled intact ciliary ganglion. P50a and P41 were derived from soluble proteins while P50b was derived from a membrane-associated protein. The proteins giving rise to P50a, P50b and P41 were also substrates for cyclic AMP-dependent protein kinase, but not for calcium and phospholipid-dependent protein kinase in vitro, suggesting that nitric oxide-responsive phosphoproteins are convergence points in information processing in vivo and their phosphorylation might represent an important mechanism in nitric oxide-mediated synaptic plasticity in ciliary ganglion.

Animals↗
PubMed1998

Alpha and beta subunits of CaM-kinase II are localized in different neurons in chick ciliary ganglion.

The ciliary ganglion of the chicken contains only two types of neurons. Using monoclonal antibodies against the alpha and the beta subunits of Ca2+/calmodulin-stimulated protein kinase II (CaMPK-II) we found that the alpha-subunit was localized to the choroid neurons while beta subunit was associated with the ciliary neurons. As both neurons receive their inputs from the oculomotor nerve, while their postganglionic axons leave via different nerves, the ciliary ganglion of the chicken is a neuronal system in which the functional differences between alpha and beta CaMPK-II homopolymers in the regulation of synaptic transmission can be investigated.

Animals↗
PubMed1996

Characterization of protein kinase and phosphatase systems in chick ciliary ganglion.

The aim of the present study was to characterize the second messenger activated protein kinase and phosphatase systems in chick ciliary ganglion using biochemical and immunochemical techniques. Using synthetic peptide substrates cyclic-AMP-, cyclic-GMP-, Ca2+/calmodulin- and Ca2+/phospholipid-dependent protein kinase activities were detected in homogenates of ciliary ganglion dissected from 15-16-day-old embryos. Autophosphorylation of the alpha and beta subunits of Ca2+/calmodulin-dependent protein kinase II in the presence of Ca2+/calmodulin or 5 mM ZnSO4 was detected by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. Protein kinase C was shown to be present using a monoclonal antibody. Two cyclic-AMP binding proteins whose molecular weights corresponded to the regulatory subunits of cyclic AMP-dependent protein kinase (RI and RII) were detected in ciliary ganglia using 8-azido-cyclic-AMP. The most heavily labelled band following incubation with [gamma-32P]ATP under most conditions had an apparent molecular weight of 65,000 which corresponds to the chicken form of myristoylated alanine-rich C kinase substrate, a known substrate of protein kinase C. Another substrate for protein kinase C was a 45,000 molecular weight protein which was tentatively identified as neuromodulin (B-50/GAP-43). Although no endogenous substrate proteins for cyclic-GMP-dependent protein kinase were detected, protein kinase A strongly labelled a 40,000 molecular weight protein. Using 32P(i)-labelled glycogen phosphorylase, protein phosphatases 1 and 2A were identified in ciliary ganglia homogenates at levels which were indistinguishable from forebrain at the same age. The major endogenous protein substrates in ciliary ganglion homogenates from 15-16-day-old embryos were also labelled to a similar extent in homogenates of ciliary ganglia from newly hatched chickens. Intact ciliary ganglia remained viable for several hours after dissection and, after incubation with 32P(i), responded to phorbol ester stimulation by an increased endogenous phosphorylation of several proteins, but especially myristoylated alanine-rich C kinase substrate. These results represent the first systematic characterization of the protein phosphorylation systems in chicken ciliary ganglion and provide a basis for future studies on the biochemical mechanisms responsible for regulating synaptic transmission in this tissue.

Animals↗
PubMed1993

Calcium/calmodulin-stimulated protein kinase II is present in primary cultures of cerebral endothelial cells.

Calcium/calmodulin-stimulated protein kinase II (CaM-PK II), a major kinase in brain, has been established to play an important role in neurotransmitter release and organization of postsynaptic receptors, and it is known to be involved in long-term potentiation and memory. Less is known about the function of this enzyme in nonneural cells. Here we report on the production, presence, and phosphorylation of the alpha-subunit of CaM-PK II in primary cultures of cerebral endothelial cells. These results raise the possibility that alpha-CaM-PK II can act as one of the key enzymes of calcium-mediated intracellular signaling in the cerebral endothelial cells and suggest that alpha-CaM-PK II may participate in such basic cellular processes as permeability in physiological and pathological conditions.

Animals↗
PubMed1993

In situ hybridization with digoxigenin labeled oligonucleotide probes: detection of CAMK-II gene expression in primary cultures of cerebral endothelial cells.

A better understanding of the regulation of gene expression under physiological and experimental conditions is one of the most important goals of today's neurobiology. In order to accomplish this task a number of in-situ hybridization methods have been elaborated. In the past few years the use of nonradioactive procedures have gained more and more space among these efforts. One of the most promising methods in this field is the application of digoxigenin-labeled probes, which have been used successfully in several laboratories. Here we present a rapid method providing good spatial resolution and low background labeling for the detection of messenger RNAs in various cell culture systems using digoxigenin-labeled probes. By using oligonucleotides complementary to the alpha and beta subunit of the calcium/calmodulin dependent protein kinase II (CAMK-II) we were able to demonstrate the presence of this enzyme in cultured cerebral endothelial cells, an enzyme that plays an important role in mediating the effects of extracellular signals.

Animals↗
PubMed1992

A chemical approach to designing Turing patterns in reaction-diffusion systems.

A systematic approach is suggested to design chemical systems capable of displaying stationary, symmetry-breaking reaction diffusion patterns (Turing structures). The technique utilizes the fact that reversible complexation of an activator species to form an unreactive, immobile complex reduces the effective diffusion constant of the activator, thereby facilitating the development of Turing patterns. The chlorine dioxide/iodine/malonic acid reaction is examined as an example, and it is suggested that a similar phenomenon may occur in some biological pattern formation processes.

Journal Article↗
PubMed1992

Regulation of transendothelial transport in the cerebral microvessels: the role of second messengers-generating systems.

Different elements of the intracellular signaling messenger systems have been detected in the course of our studies in the cerebral endothelial cells. It has been shown that the synthesizing enzymes of and substrate proteins for the second messenger molecules are present in the cerebral endothelial cells, and their activity and/or amount can change in pathological circumstances, i.e., during the formation of brain oedema. Pharmacological treatments interfering with the second messenger systems proved to be effective in the prevention of brain oedema formation.

Adenylyl Cyclases↗