Nifedipine blocks apamin-induced bursting activity in nigral dopamine-containing neurons.

Intrinsic sinusoidal oscillations in membrane potential, characteristic of nigral dopamine cells, are converted to plateau potentials following application of apamin, a potent antagonist of SK-type Ca2+-activated K+ channels. Blockade of these channels also changes neuronal firing pattern from a single-spike pacemaker discharge to a multiple spike bursting pattern. Nifedipine, a selective antagonist of L-type Ca2+ channels, blocks plateau potential generation; however, its effects on firing pattern have yet to be determined. In the present study, extracellular single unit recording techniques were used in conjunction with a brain slice preparation to determine whether nifedipine, in a concentration known to block plateau potential generation, also affects bursting activity. Nifedipine (30 microM) was equipotent in inhibiting the firing rate of control (51.2+/-10.8%) and apamin-treated (44.9+/-5.4%) neurons. Slow firing neurons (<2 Hz) were particularly sensitive to the inhibitory effects of the drug. Apamin-induced bursting was completely suppressed by nifedipine and accompanied by a significant increase in the regularity of firing. By contrast, pacemaker-like activity exhibited by control neurons was unaffected by the drug. These data demonstrate that the intrinsic plateau properties exhibited by DA neurons are responsible for the generation of phasic activity induced following blockade of apamin-sensitive Ca2+-activated K+ channels and provide further support for the involvement of an L-type Ca2+ conductance in mediating this type of activity.

Apamin blocks the direct relaxant effect of melatonin on rat ileal smooth muscle.

The present study investigated the mechanisms of melatonin-induced inhibition of the ileal smooth muscle contraction. Rat isolated ileal smooth muscle strips were stimulated in an organ bath using carbachol (CAR) or potassium chloride (KCl) depolarization. Under these conditions, melatonin produced a concentration-dependent inhibition of muscle contraction (mean inhibitory concentration, IC50: 17.3 x 10(-6) M), which was not blocked by either tetrodotoxin (10(-6) M), hexamethonium (10(-4) M), or phentolamine (10(-6) M). The inhibitory effect of melatonin during CAR stimulation was blocked in a concentration-dependent manner by the presence of apamin (4.8 x 10(-9) M), a K(+)-channel blocker. By contrast, other K(+)-channel blockers such as 4-aminopyridine (10(-4) M to 5 x 10(-3) M), tetraethylammonium (10(-4) to 10(-1) M), and glibenclamide (10(-5) M) were ineffective. Additionally, the Ca(2+)-channel antagonists nitrendipine (IC50: 2.4 x 10(-9) M) and verapamil (IC50: 1.1 x 10(-7) M) also blocked the inhibitory action of melatonin. These results suggest that melatonin may interact with an apamin-sensitive, possibly Ca(2+)-activated, K+ channel and thus cause an inhibition of ileal smooth muscle contractions.

Characterization of PO1, a new peptide ligand of the apamin-sensitive Ca2+ activated K+ channel.

A new peptide ligand of the small conductance Ca2+ activated K+ channels has been purified from the venom (obtained by manual rather than electrical stimulation of the scorpion Androctonus mauretanicus mauretanicus), by following the inhibition of the 125I-apamin binding to its receptor on rat brain synaptosomes. Only one step on a C18 reversed-phase high-performance liquid chromatography column was necessary to obtain PO1. Its K0.5 for the apamin binding site was 100 nM. The amino acid sequence of PO1 is different from those of leiurotoxin and PO5. For the first time the same peptide was also purified from the venoms of two other species of North African scorpions, Androctonus australis and Buthus occitanus tunetanus. PO1 was chemically synthesized by the solid-phase technique and fully characterized. A model of PO1 was constructed by amino acid replacement using PO5 nuclear magnetic resonance studies as the starting model. Structure-activity relationships between these toxins and their receptor are discussed.

Characterization of the apamin- and L-nitroarginine-resistant NANC inhibitory transmission to the circular muscle of guinea-pig colon.

1. The aim of this study was a pharmacological characterization of the multiple NANC inhibitory transmission systems producing relaxation of the circular muscle of guinea-pig proximal colon. In the presence of atropine (1 microM), guanethidine (3 microM) and of the tachykinin NK1 and NK2 receptor antagonists, SR 140333 (0.3 microM) and MEN 10627 (1 microM), respectively, electrical field stimulation (EFS) produced a frequency-dependent (0.1-3 Hz) relaxation. During a cumulative frequency-response curve, the maximal relaxant effect was produced at 3 Hz and approached the maximal relaxation to 1 microM isoprenaline. In the presence of both apamin (0.3 microM) and L-nitroarginine (L-NOARG, 100 microM), EFS failed to evoke relaxation up to 1 Hz; at 1-10 Hz, a slowly developing relaxation ensured which approached 50% of the Emax to isoprenaline. The EFS-evoked NANC relaxation, either in the presence or absence of apamin and L-NOARG, was unaffected by in vitro capsaicin pretreatment (10 microM for 15 min). 2. Three protocols of EFS were developed for further pharmacological analysis: (a) EFS at 1 Hz for 5 s in the presence of L-NOARG, producing a transient fast apamin-sensitive relaxation; (b) EFS at 1 Hz for 5 s in the presence of apamin, producing a transient fast L-NOARG-sensitive relaxation; and (c) EFS at 10 Hz for 5 s in the presence of both apamin and L-NOARG, producing a transient but slowly developing and more sustained relaxation. 3. The neutral endopeptidase inhibitor, thiorphan (1-10 microM), enhanced and prolonged the apamin- and L-NOARG-resistant NANC relaxation produced by EFS at 10 Hz, without affecting that evoked at 1 Hz in the presence of apamin or L-NOARG. The angiotensin converting enzyme inhibitor, captopril (1-10 microM) was without effect. 4. The cAMP analogue inhibitor of protein kinase A, Rp-cAMPs (100-300 microM) significantly reduced and shortened the NANC relaxation produced by 10 Hz EFS in the presence of L-NOARG without affecting that produced by 1 Hz EFS in the presence of apamin or L-NOARG. 5. The inhibitor of sarcoplasmic reticulum Ca-ATPase, cyclopiazonic acid (CPA, 3-10 microM for 60 min) abolished the 1 Hz EFS-induced relaxation in the presence of L-NOARG, and greatly inhibited that produced by 10 Hz EFS in the presence of both apamin and L-NOARG. The relaxation produced by 1 Hz EFS in the presence of apamin was inhibited by about 32% at 10 microM only. 6. Nifedipine (1 microM) did not affect the EFS-induced NANC relaxations. In the presence of nifedipine, tetraethylammonium (TEA, 1 mM) enhanced the 1 Hz EFS-induced relaxation in the presence of L-NOARG (158% of control) and that produced by 10 Hz EFS in the presence of apamin and L-NOARG (215% of control) while that evoked by 1 Hz EFS in the presence of apamin was slightly affected (109% of control). 7. In the presence of atropine, guanethidine, SR 140333 and MEN 10627, bath application of human vasoactive intestinal polypeptide (VIP, 0.1 nM-10 nM) produced a concentration-dependent, slowly developing relaxation of colonic strips. The relaxation to VIP was unaffected by apamin (0.3 microM), L-NOARG (100 microM), nifedipine (1 microM) or nifedipine plus TEA (1 mM); it was inhibited by CPA (10 microM) and Rp-cAMPs (100 microM) and was potentiated by thiorphan (10 microM). 8. The putative VIP receptor antagonist, VIP(10-28) (10 microM) did not affect the VIP-induced relaxation nor the NANC relaxation to 10 Hz EFS in the presence of apamin and L-NOARG. 9. The present findings provide evidence that three distinct NANC inhibitory mechanisms mediate relaxation of the circular muscle of the guinea-pig proximal colon. The first system provides a fast relaxation in response to low frequency of stimulation and may involve the action of a transmitter(s) (possibly ATP) which mobilizes intracellular Ca2+ from sarcoplasmic reticulum leading to the activation of apamin-sensitive K+ channels. The second system likewise provides a fast relaxation of the colon in

Pharmacology of the high-affinity apamin receptor in rabbit heart.

UNLABELLED: Apamin is a potent blocker of calcium-activated small conductance potassium (SK) channels in neurons, liver, skeletal muscle and ileum smooth muscle, but not in cardiac muscle. Cardiac muscle is devoid of SK channels; however, in isolated, single ventricular myocytes apamin is an extremely potent blocker of the L-type calcium current, and the anti-arrhythmic drug quinidine reverses apamin block. OBJECTIVE: To characterize the receptor binding properties and pharmacology of the apamin receptor in heart. METHODS: The binding properties of the apamin receptor were determined by rapid filtration of purified rabbit heart membranes. RESULTS: Monoiodinated apamin binds to a labile, membrane-bound protein in heart membranes at a single, high-affinity site (KD = 8.07 +/- 2.14 pM and Bmax = 686 +/- 167 fmoles/mg protein, significant run test at P = 0.05 for a one site fit). 125I-apamin binding is dose-dependently inhibited by apamin, scyllatoxin, quinidine, amiloride, as well as a variety of di- and trivalent cations that are classical blockers of L-type calcium channels (e.g. Co2+, Cd2+, Mn2+, La3+, Gd3+). The cardiac apamin receptor is also critically dependent upon pH, temperature and KCl, and co-purifies in the same membrane fraction as L-type cardiac Ca2+ channels. CONCLUSIONS: The apamin receptor in rabbit heart P2 membranes has pharmacological and biochemical properties in common with both an SK channel and an L-type Ca2+ channel.

Determination of the three-dimensional solution structure of scyllatoxin by 1H nuclear magnetic resonance.

The three-dimensional solution structure of Scyllatoxin (leiurotoxin I) a venom peptide from the scorpion Leiurus quinquestriatus hebraeus was determined at 1 A resolution by homonuclear proton n.m.r. methods at 500 MHz. Data analysis and structure calculation followed conventional protocols inherent to DIANA and related programs with two exceptions. First, distance constraints were obtained from two-dimensional nuclear Overhauser spectra by a previously described partial relaxation matrix approach. Second, since the pairing pattern of the six cysteine residues was not established a priori, the unequivocal assignment of the disulfide bridges was achieved exclusively from the n.m.r. data by a statistical analysis of preliminary DIANA structures. In the final calculation we used 227 upper distance constraints, 67 torsional constraints from vicinal coupling constants and ten stereospecific assignments of beta-methylene protons. Scyllatoxin folds into a compact ellipsoidal shape. An alpha-helix (defined with 0.24 A resolution) spanning 2.5 turns from Leu5 till Ser14 is stabilized by Cys8-Cys26 and Cys12-Cys28 disulfide bridges to the carboxy-terminal strand of an anti-parallel beta-sheet. The antiparallel beta-sheet (defined at 0.66 A resolution) extends from Leu18 to Val29 with a tight turn at Gly23-Asp24 and displays a right-handed twist. Scyllatoxin competes with the toxins apamin from Apis mellifera mellifera and P05 from Androctonus mauretanicus mauretanicus for the same or similar high conductance calcium-activated potassium channels. Consideration of presently known biological activities and three-dimensional structures of these toxins suggest a different toxin-receptor interaction of scyllatoxin as compared to apamin and P05.

Structure-activity relationship study of a scorpion toxin with high affinity for apamin-sensitive potassium channels by means of the solution structure of analogues.

Scorpion venoms contain numerous toxic polypeptides displaying various pharmacological activities. These toxins interact with ion channels of excitable membranes. Long toxins (60-70 amino acids) are known to interact with sodium channels, whereas most of the short toxins (31-37 amino acids) found their toxicity in modifying the potassium channel functions. A family of short scorpion toxins are known to interact specifically with apamin-sensitive calcium-activated potassium channels. Structure-activity relationship studies of these toxins have demonstrated that a short region located on the solvent-exposed side of an alpha-helix is involved in the interaction with their receptor. Two positions, i.e. residues 6 and 7 in the sequence, are essential for the full activity of these molecules. We have synthesized analogues of these toxins and demonstrated that the three-dimensional structure is not affected by these mutations, and thus that the observed variations of activity are only due to the chemical function carried by the side chain. This interaction between the toxins and their receptor is thus purely electrostatic.

Evidence that Ca(2+)-activated K+ channels participate in the regulation of pituitary prolactin secretion.

We evaluated the role of Ca(2+)-activated K+ channels in the regulation of prolactin (PRL) secretion with a perifusion system using acutely dispersed rat anterior pituitary cells. Apamin, which blocks Ca(2+)-activated K+ channels, induced PRL secretion in a dose-dependent fashion between 1 and 300 nM (r = 0.99, P < 0.01). Charybdotoxin, another Ca(2+)-activated K+ channel-blocker, also induced PRL secretion at 20 nM concentration. These were not non-specific toxic effects, since stimulation of PRL secretion by 10 nM thyrotropin-releasing hormone (TRH) was not different before and after applying the channel-blockers. Both 10 microM dopamine and 2 microM nifedipine significantly, but incompletely, depressed PRL secretion induced by 100 nM apamin; 10 microM dopamine completely blocked PRL secretion induced by 20 nM charybdotoxin. Our data indicate that Ca(2+)-activated K+ channels may play an important role in the regulation of PRL secretion.

Leiurotoxin I, a scorpion toxin specific for Ca(2+)-activated K+ channels. Structure-activity analysis using synthetic analogs.

Recently, we reported a structure-activity relationship study on P05, a novel leiurotoxin I-like scorpion toxin which is selective for the apamin-sensitive Ca(2+)-activated K+ channel [Sabatier et al. (1993) Biochemistry 32, 2763-2770]. Arg6, Arg7 and C-terminal His31 appeared to be key residues for P05 biological activity. Owing to the high sequence identity between P05 and leiurotoxin I (87%), several analogs of leiurotoxin I (Lei-NH2) with point mutations at these positions were designed and chemically synthesized using an optimized solid-phase technique. The synthesized peptides were [L6]Lei-NH2, [R7]Lei-NH2, Lei-OH and [R7]Lei-OH, as well as fragment [R7,Abu8]N4-S11-NH2. A chimeric analog ([M22,K24,R27]Lei-NH2), which possesses part of the iberiotoxin C-terminus, was also constructed. Circular dichroism analyses of these analogs, in agreement with their structural models obtained by molecular dynamics, showed that the point mutations did not significantly affect the overall secondary structures, as compared to natural Lei-NH2. All the peptides and natural toxins were compared in vitro for their capacity to inhibit binding of [125I]-apamin to rat brain synaptosomes, and in vivo for their specific neurotoxicity in mice. The Arg6 residue was essential for high biological activity of leiurotoxin I. Further, substitution of Met7 in the natural toxin by Arg7, or C-terminal amidation of His31, greatly increased affinity for the apamin receptor but did not significantly affect toxin neurotoxicity. Remarkably, the chimeric analog [M22,K24,R27]Lei-NH2 was found to retain leiurotoxin I-like activity, thus indicating that the negatively charged residues Asp24 and Glu27 (and Ile22) are not directly involved in the high toxin bioactivity. However, the chimeric molecule had no iberiotoxin-like effect on rat muscular maxi-K+ channels incorporated in lipid bilayers.

Solution structure of a core peptide derived from scyllatoxin.

An analysis of the sequences of scyllatoxin and charybdotoxin suggested that it would be possible to design a core peptide sequence which would still fold to give the beta-hairpin and helix seen in the toxins, but which would eliminate one disulfide and connecting residues. The core sequence was modeled, then synthesized and purified. The cysteines oxidize in air to give the same disulfide pairings as seen in the parent toxins as the major product. The three-dimensional structure of the core sequence peptide, termed Max, was determined using proton NMR spectroscopy and found to be identical in secondary structure to the toxins. However differences were found in the relative orientation of the beta-hairpin and helix. The use of this structural motif, found in many insect toxins, as a disulfide framework for exploring sequence/structure/activity relationships is discussed.

Dequalinium: a potent inhibitor of apamin-sensitive K+ channels in hepatocytes and of nicotinic responses in skeletal muscle.

The bisquaternary compound dequalinium has been tested for its ability to inhibit the loss of K+ which angiotensin II causes in guinea-pig hepatocytes and which occurs through apamin-sensitive Ca(2+)-activated K+ (SKCa) channels. Dequalinium blocked angiotensin II-evoked K+ loss with an IC50 of 1.5 +/- 0.1 microM and also inhibited 125I-monoiodoapamin binding with a KI of 1.1 +/- 0.1 microM. It is the most active non-peptide SKCa blocker so far described. The neuromuscular blocking agent vecuronium was also tested, and proved to be considerably less effective (IC50, 4.5 +/- 0.3 microM; KI, 3.6 +/- 0.5 microM). Dequalinium was also examined for its actions at nicotinic receptors in skeletal muscle and was found to be a potent, non-competitive antagonist of carbachol contractions of the frog rectus abdominis. In the frog cutaneous pectoris muscle, end-plate depolarizations induced by carbachol became smaller and more transient in the presence of dequalinium at 10 nM. However, contractions of the frog sartorius and rat diaphragm in response to nerve stimulation were inhibited only by concentrations > 1 microM. These apparently discrepant effects of dequalinium on nicotinic responses could be explained either by open channel block of slow onset or by 'stabilization' of the desensitized state of the receptor. The potency of dequalinium will make it a useful agent for the study of nicotinic receptors as well as of SKCa channels.

Characterization and partial purification from pheochromocytoma cells of an endogenous equivalent of scyllatoxin, a scorpion toxin which blocks small conductance Ca(2+)-activated K+ channels.

This work describes the partial purification of a heat-stable peptide which has the same properties as the scorpion toxin, scyllatoxin, a specific blocker of one class of Ca(2+)-activated K+ channels: (i) it competes with [125I]apamin for binding to the same site, (ii) like apamin and scyllatoxin, it blocks the after-potential hyperpolarization in skeletal muscle cells in culture, (iii) like apamin and scyllatoxin, it contracts guinea-pig taenia coli relaxed by epinephrine, (iv) it cross-reacts with antibodies raised against scyllatoxin but not with antibodies raised against apamin.

Effect of charybdotoxin and leiurotoxin I on potassium currents in bullfrog sympathetic ganglion and hippocampal neurons.

The effects of charybdotoxin and leiurotoxin I were examined on several classes of K+ currents in bullfrog sympathetic ganglion and hippocampal CA1 pyramidal neurons. Highly purified preparations of charybdotoxin selectively blocked a large voltage- and Ca(2+)-dependent K+ current (IC) responsible for action potential repolarization (IC50 = 6 nM) while leiurotoxin I selectively blocked a small Ca(2+)-dependent K+ conductance (IAHP) responsible for the slow afterhyperpolarization following an action potential (IC50 = 7.5 nM) in bullfrog sympathetic ganglion neurons. Neither of the toxins had significant effects on other K+ currents (M-current [IM], A-current [IA] and the delayed rectifier [IK]) present in these cells. Leiurotoxin I at a concentration of 20 nM had no detectable effect on currents in hippocampal CA1 pyramidal neurons. This lack of effect on IAHP in central neurons suggests that the channels underlying slow AHPs in those neurons are pharmacologically distinct from analogous channels in peripheral neurons.

Scyllatoxin, a blocker of Ca(2+)-activated K+ channels: structure-function relationships and brain localization of the binding sites.

Chemical modifications of scyllatoxin (leiurustoxin I) have shown that two arginines in the sequence, Arg6 and Arg13, are essential both for binding to the Ca(2+)-activated K+ channel protein and for the functional effect of the toxin. His31 is important both for the binding activity of the toxin and for the induction of contractions on taenia coli. However, although its iodination drastically decreases the toxin activity, it does not abolish it. Chemical modification of lysine residues or of Glu27 does not significantly alter toxin binding, but it drastically decreases potency with respect to contraction of taenia coli. The same observation has been made after chemical modification of the lysine residues. The brain distribution of scyllatoxin binding sites has been analyzed by quantitative autoradiographic analysis. It indicates that apamin (a bee venom toxin) binding sites are colocalized with scyllatoxin binding sites. The results are consonant with the presence of apamin/scyllatoxin binding sites associated with Ca(2+)-activated K+ channels. High-affinity binding sites for apamin can be associated with very-high-affinity (less than 70 pM), high-affinity (approximately 100-500 pM), or moderate-affinity (greater than 800 pM) binding sites for scyllatoxin.

Characterization and regulation of the expression of scyllatoxin (Leiurotoxin I) receptors in the human neuroblastoma cell line NB-OK 1.

125I-[Tyr2]scyllatoxin allowed to label a single class of high-affinity receptors in membranes from the human neuroblastoma cell line NB-OK 1. The Kd of these receptors was 60 pM for scyllatoxin (Leiurotoxin I) and 20 pM for apamin and the Bmax was low (3.8 fmol/mg membrane protein). K+ increased toxin binding at low concentrations but exerted opposite effects at high concentrations. Ca2+, guanidinium and Na+ exerted only inhibitory effects on binding. Scyllatoxin binding sites were overexpressed 2.5-fold after a 24-h cell pretreatment with 2 mM butyrate. This effect was suppressed by cycloheximide.

Effect of microinjections of apamin into the A10 dopamine region of rats: a behavioral and neurochemical analysis.

Hyperpolarization of dopamine neurons by activation of D2 and gamma-aminobutyric acidB receptors involves an increased conductance of K+ ions. Apamin, a blocker of Ca2(+)-activated K+ channels, has been reported to increase activity of dopamine neurons. Increased activity of the mesolimbic dopamine system is associated with increased motor activity. Thus, we investigated the behavioral and neurochemical effects of acute and daily microinjections of apamin into the A10 region of the rat. Apamin increased motor activity in a dose-dependent manner, and this effect was blocked by pretreatment with 0.1 mg/kg haloperidol. In postmortem analysis, 6.0 pmol of apamin significantly increased the levels of dihydroxyphenylacetic acid in the A10 region and of dihydroxyphenylacetic acid and homovanillic acid in the nucleus accumbens, and 2.0 pmol of apamin significantly increased the level of dopamine in the prefrontal cortex. In vivo dialysis in the nucleus accumbens of freely moving rats revealed that apamin elevated extracellular dopamine metabolites. Rats receiving daily microinjections of apamin into the A10 region did not exhibit an augmentation in motor activity, suggesting that rats did not become sensitized to chronic treatment. These data are discussed in terms of the role of apamin-sensitive dopamine mechanisms in motor behavior and sensitization of these motor behaviors.

Leiurotoxin I (scyllatoxin), a peptide ligand for Ca2(+)-activated K+ channels. Chemical synthesis, radiolabeling, and receptor characterization.

Leiurotoxin I (scyllatoxin) is a 31-amino acid polypeptide from the venom of the scorpion Leiurus quinquestriatus hebraeus which has been previously isolated and sequenced by others. This paper reports (i) the total synthesis of this scorpion neurotoxin as well as some aspects of its structure-function relationships; (ii) the synthesis of the analog [Tyr2]leiurotoxin I (scyllatoxin) that has been monoiodinated at high specific radioactivity (2000 Ci/mmol) and has served for the characterization of the properties of 125I-[Tyr2]leiurotoxin I binding sites (Kd = 80 pM, molecular mass of 27 and 57 kDa for two polypeptides in the leiurotoxin I binding protein); (iii) the similarity of physiological actions between leiurotoxin I and apamin. Both toxins contract Taenia coli previously relaxed with epinephrine, both toxins block the after-hyperpolarization due to Ca2(+)-activated K+ channel activity in muscle cells in culture; (iv) the probable identity of binding sites for apamin and leiurotoxin I. In spite of a different chemical structure apamin competitively inhibits 125I-[Tyr2] leiurotoxin I binding and vice versa. Moreover, the peculiar effects of K+ on 125I-[Tyr2]leiurotoxin I binding are identical to those previously observed for 125I-apamin binding.

Purification and characterization of a unique, potent inhibitor of apamin binding from Leiurus quinquestriatus hebraeus venom.

An inhibitor of apamin binding has been purified to homogeneity in three chromatographic steps from the venom of the scorpion, Leiurus quinquestriatus hebraeus. The inhibitor, which we have named leiurotoxin I, represents less than 0.02% of the venom protein. It is a 3.4-kDa peptide with little structural homology to apamin although it has some homology to other scorpion toxins such as charybdotoxin, noxiustoxin, and neurotoxin P2. Leiurotoxin I completely inhibits 125I-apamin binding to rat brain synaptosomal membranes (Ki = 75 pM). Thus, it is 10-20-fold less potent than apamin. Leiurotoxin I is not a strictly competitive inhibitor of this binding reaction. Like apamin, leiurotoxin I blocks the epinephrine-induced relaxation of guinea pig teniae coli (ED50 = 6.5 nM), while having no effect on the rate or force of contraction in guinea pig atria or rabbit portal vein preparations. Thus, leiurotoxin I of scorpion venom and apamin of honeybee venom demonstrate similar activities in a variety of tissues, yet are structurally unrelated peptides. These two peptides should be useful in elucidating the role of the small conductance, Ca2+-activated K+ channels in different tissues.