Calcium Antagonistic Properties Research Articles

Chalepin and Chalepensin: Occurrence, Biosynthesis and Therapeutic Potential.

Dihydrofuranocoumarin, chalepin (1) and furanocoumarin, chalepensin (2) are 3-prenylated bioactive coumarins, first isolated from the well-known medicinal plant Ruta chalepensis L. (Fam: Rutaceae) but also distributed in various species of the genera Boenminghausenia, Clausena and Ruta. The distribution of these compounds appears to be restricted to the plants of the family Rutaceae. To date, there have been a considerable number of bioactivity studies performed on coumarins 1 and 2, which include their anticancer, antidiabetic, antifertility, antimicrobial, antiplatelet aggregation, antiprotozoal, antiviral and calcium antagonistic properties. This review article presents a critical appraisal of publications on bioactivity of these 3-prenylated coumarins in the light of their feasibility as novel therapeutic agents and investigate their natural distribution in the plant kingdom, as well as a plausible biosynthetic route.

Hibiscus Sabdariffa L. Flowers and Olea Europea L. Leaves Extract-Based Formulation for Hypertension Care: In Vitro Efficacy and Toxicological Profile.

Olea europaea L. leaves extract (Oe) and Hybiscus sabdariffa L. flowers extract (Hs) have calcium antagonistic properties. Aim of this work was to study the cardiovascular effects of Pres Phytum(®), a nutraceutical formulation containing a mixture of the two extracts and the excipients, and investigate its possible off-target effects, using in vitro biological assays on guinea pig isolated organs. Cardiovascular effects were assessed using guinea pig atria and aorta. The effects of Pres Phytum on spontaneous gastrointestinal, urinary, and respiratory tracts smooth muscle contractility were evaluated. Pres Phytum exerted a vasorelaxant effect (IC50 = 2.38 mg/mL) and a negative chronotropic effect (IC50 = 1.04 mg/mL) at concentrations lower than those producing smooth muscle spontaneous contractility alterations in the other organs. Compared to Pres Phytum, the mixture did not exert negative inotropic activity, while it maintained a negative chronotropic efficacy (IC50 = 1.04 mg/mL). These experimental data suggest a possible nutraceutical use of this food supplement for the management of preclinical hypertension.

Levetiracetam, Calcium Antagonism, and Bipolar Disorder.

Hyperactive intracellular calcium ion (Ca) signaling in peripheral cells has been a reliable finding in bipolar disorder. Some established mood stabilizing medications, such as lithium and carbamazepine, have been found to normalize elevated intracellular Ca concentrations ([Ca]i) in platelets and lymphocytes from bipolar disorder patients, and some medications the primary effect of which is to attenuate increased [Ca]i have been reported to have mood stabilizing properties.Hyperactive intracellular Ca signaling has also been implicated in epilepsy, and some anticonvulsants have calcium antagonist properties. This study demonstrated that levetiracetam, an anticonvulsant that has been shown to block N and P/Q-type calcium channels in animal studies does not alter elevated [Ca]i in blood platelets of patients with bipolar disorder. Review of published clinical trials revealed no controlled evidence of efficacy as a mood stabilizer.This study underscores the possibility that pharmacologic actions of a medication in animals and normal subjects may not necessarily predict its pharmacologic or clinical effects in actual patients. Effects of treatments on pathophysiology that is demonstrated in clinical subtypes may be more likely to predict effectiveness in those subtypes than choosing medications based on structural similarities to established treatments.

Synthesis and studies of calcium channel blocking and antioxidant activities of novel 4-pyridinium and/or N-propargyl substituted 1,4-dihydropyridine derivatives

Abstract The novel 1,4-dihydropyridine derivatives containing the cationic pyridine moiety at the position 4, and the N -propargyl group as a substituent at position 1 of the 1,4-DHP cycle were designed, synthesised, and assessed in biological tests. Among all the novel compounds, the 4-( N -dodecyl) pyridinium group-containing compounds 11 (without the N -propargyl group) and 12 (with the N -propargyl group) demonstrated the highest calcium antagonistic properties against neuroblastoma SH-SY5Y (IC 50 about 5–14 μM) and the vascular smooth muscle A7r5 cell (IC 50 – 0.6–0.7 μM) lines, indicating that they predominantly target the L-type calcium channels. These compounds showed a slight total antioxidant activity. At concentrations close to those of L-type calcium channel blocking ones, compound 12 did not affect mitochondrial functioning; also, no toxicity was obtained in vivo. The N -propargyl group as a substituent at position 1 of the 1,4-DHP cycle did not essentially influence the compounds’ activity. The 4-( N -dodecyl) pyridinium moiety-containing compounds can be considered as prototype molecules for further chemical modifications and studies as cardioprotective/neuroprotective agents.

The inhibitory effects of m-nisoldipine on the 5-hydroxytryptamine-induced proliferation of pulmonary artery smooth muscle cells via Ca2+ antagonism and antioxidant mechanisms

The excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) plays a critical role in the development of pulmonary arterial hypertension. Recent studies indicate that Ca2+ and reactive oxygen species are critically involved in the process of smooth muscle cell proliferation stimulated by mitogens, such as 5-hydroxytryptamine (5-HT). Because m-nisoldipine, a Ca2+ channel blocker of the dihydropyridine class, possesses some calcium antagonistic and antioxidant properties, we investigated the effect of m-nisoldipine on PASMC proliferation. The results indicated that m-nisoldipine inhibited 5-HT-induced PASMC proliferation, evaluated by BrdU incorporation and the MTT assay, and this effect was associated with a decreased expression of proliferating cell nuclear antigen (PCNA). Flow cytometry analysis showed that m-nisoldipine blocked 5-HT-induced cell-cycle progression by arresting the cells in the G0/G1 phase. Next, the production of reactive oxygen species and the levels of [Ca2+]i in PASMCs were measured by laser scanning confocal microscopy; m-nisoldipine pretreatment attenuated the [Ca2+]i elevation and the production of reactive oxygen species induced by 5-HT. In addition, m-nisoldipine significantly decreased the 5-HT-induced activation of extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal kinase (JNK) and the subsequent c-fos and c-jun mRNA expression. Meanwhile, results also showed that N-acetylcysteine (a reactive oxygen species scavenger) suppressed the proliferation and the ERK1/2 and JNK activation induced by 5-HT. In summary, this study demonstrated that m-nisoldipine effectively suppressed the 5-HT-induced PASMC proliferation, ERK1/2 and JNK activation and subsequent c-fos and c-jun mRNA expression, all of which might be associated with the Ca2+ antagonistic and antioxidant properties of m-nisoldipine.

P.3.015 Hyperhomocysteinemia, methylenetetrahydrofolate reductase 677C→T polymorphism and psychiatric disorders in children

The novel 1,4-dihydropyridine derivatives containing the cationic pyridine moiety at the position 4, and the N-propargyl group as a substituent at position 1 of the 1,4-DHP cycle were designed, synthesised, and assessed in biological tests. Among all the novel compounds, the 4-(N-dodecyl) pyridinium group-containing compounds 11 (without the N-propargyl group) and 12 (with the N-propargyl group) demonstrated the highest calcium antagonistic properties against neuroblastoma SH-SY5Y (IC50 about 5–14 μM) and the vascular smooth muscle A7r5 cell (IC50 – 0.6–0.7 μM) lines, indicating that they predominantly target the L-type calcium channels. These compounds showed a slight total antioxidant activity. At concentrations close to those of L-type calcium channel blocking ones, compound 12 did not affect mitochondrial functioning; also, no toxicity was obtained in vivo. The N-propargyl group as a substituent at position 1 of the 1,4-DHP cycle did not essentially influence the compounds’ activity. The 4-(N-dodecyl) pyridinium moiety-containing compounds can be considered as prototype molecules for further chemical modifications and studies as cardioprotective/neuroprotective agents.

Calcium antagonistic activity of Bacopa monniera in guinea-pig trachea

Objective:To demonstrate the calcium antagonistic property of ethanol extract of Bacopa monniera in guinea-pig trachea.Materials and Methods:The dose response curves of CaCl2 (1 × 10-5 to 1 × 10-1 M) were constructed in the absence and presence of ethanol extract of Bacopa monniera (100, 500 and 700 μg/ml) or nifedipine (1 × 10-6 M) in guinea-pig trachea in calcium free high K+-MOPS-PSS (3-(N-morpholino)-propanesulphonic acid physiological salt solution). The data was analyzed by ANOVA followed by least significant difference test or by Student's ‘t’ test for unequal variance when appropriate. A probability of at least P < 0.05 was considered statistically significant.Results:The plant extract (500 and 700 μg/ml) significantly (P < 0.05) depressed and shifted the calcium concentration-response curves (1 × 10-3- 1 × 10-1 M) to rightward similar to that of nifedipine.Conclusions:Bacopa monniera extract exhibited calcium channel blocking activity in guinea-pig tracheal smooth muscles that may rationalize its relaxant action on guinea-pig trachea and its traditional use in respiratory disorders.

Synthesis and Biological Activity of Thienodiltiazem in Isolated Heart Muscle Preparations of Guinea Pigs

The new compound thienodiltiazem was synthesized and investigated regarding structure-activity relations and calcium antagonistic properties. Isometric contraction force was measured in guinea-pig papillary muscles and chronotropic activity was studied in right atria of guinea pigs. Compared to the parent drug diltiazem (CAS 42399-41-7) the thieno derivative had a more potent negative chronotropic effect on spontaneously beating right atria and a more potent inotropic effect on papillary muscles. The negative inotropic action was reversed by increasing the extracellular calcium concentration.

Dilazep: an inhibitor of adenosine uptake with intrinsic calcium antagonistic properties.

Concentrations of dilazep which were ineffective in altering the muscular tone of the guinea-pig taenia caeci (0.03, 0.3 microM) or the phasic mechanical activity of the rabbit proximal ileum (0.03 microM) markedly potentiated the inhibitory action of adenosine on both these parameters. Dilazep, 0.3 microM or greater, dose-dependently inhibited the mechanical activity of the proximal ileum. This inhibitory action was probably mediated by more than one mechanism, as shown by the fact that theophylline (50, 100 microM) antagonized the effect at lower dilazep concentrations (up to 3 microM) leaving essentially unchanged the response to higher concentrations (6, 10 microM). Similarly, the responses to low doses of dilazep were reduced after desensitization of the organ to adenosine, whilst the responses to higher doses were unaffected by this procedure. In a Ca2+-free, high-K+ medium, dilazep (1-10 microM) caused a parallel shift to the right of the Ca2+-induced contractions of the guinea-pig taenia caeci. Adenosine showed only slight Ca2+-antagonistic properties within the mM range of concentrations. These findings suggest that, at the higher concentration tested, dilazep exhibits Ca2+-antagonistic properties unrelated to its adenosine-mediated mode of action.

Calcium Antagonist Properties of Cinnarizine, Trifluoperazine and Verapamil in Guinea-pig Normal and Skinned Trachealis Muscle

In guinea-pig trachealis, depolarized by a K(+)-rich medium, Ca2+ (0.01-10 mM) caused concentration-related spasm. Verapamil (0.5-5 microM), cinnarizine (10-100 microM) and trifluoperazine (16-160 microM) each produced concentration-dependent antagonism of Ca2+ characterized by a rightward and downward displacement of the log concentration-effect curve for Ca2+. The rank order of potencies of these antagonists, measured as the IC75 against Ca2+ (10 mM)-induced contraction of depolarized trachea, was verapamil (5.6 microM) greater than cinnarizine (59 microM) greater than trifluoperazine (91 microM). In skinned trachea, verapamil in concentrations up to 100 microM did not modify the concentration-effect curve for Ca2+. In contrast, cinnarizine (59-177 microM) diminished the sensitivity and trifluoperazine (273 microM) decreased the responsiveness of the tissue to Ca2+. In skinned trachea, trifluoperazine (91 microM) produced greater inhibition of Ca2+ (10 microM)-induced contraction after 120 min than after 30 min of incubation. Verapamil (100 microM) and cinnarizine (177 microM) were devoid of inhibitory effect against the 10 microM Ca2+ standard. In skinned trachea, changes in the Ca2+ concentration-effect curve produced by cinnarizine (177 microM) were reversed after washout whilst those induced by trifluoperazine (273 microM) persisted. It is concluded that distinct differences exist between the three calcium antagonists examined. The action of verapamil is restricted to the plasmalemma. That of cinnarizine and trifluoperazine is exerted both on the plasma membrane and upon the intracellular contractile machinery.

ChemInform Abstract: Xanthone 1,4-Dihydropyridine Derivatives with a Potent Selective Bradycardic Effect.

A series of xanthone 1,4-dihydropyridine derivatives were prepared. The compounds were evaluated for inotropic, chronotropic and calcium antagonistic properties. The tested compounds are weak calcium antagonists but exert potent selective bradycardic effects.

ChemInform Abstract: 4-Heterotricyclic Substituted 1,4-Dihydropyridines with a Potent Selective Bradycardic Effect.

A series of 4-heterotricyclic substituted 1, 4-dihydropyridines were prepared. The compounds were evaluated for inotropic, chronotropic and calcium antagonist properties. The tested compounds are fairly good as calcium antagonists, but exert potent selective bradycardic effects

ChemInform Abstract: SAR Studies in the Field of Ca-Antagonists: 2-Substituted 1,4- Dihydropyridines with a Xanthone Backbone.

A series of 2-substituted 1,4-dihydropyridines with a xanthone backbone was prepared. The compounds were evaluated for inotropic, chronotropic and calcium antagonist properties.

ChemInform Abstract: Synthesis and Pharmacological Characterization of Some Benzazepinone Derivatives.

A series of some benzazepinone derivatives were prepared. The compounds were evaluated for inotropic, chronotropic and calcium antagonist properties. For the compounds bearing well-known alpha-blocking fragments, the antagonist potency at postsynaptic alpha-adrenoceptors was assessed. The new compounds were less effective as bradycardic agents and calcium antagonists but show remarkable negative inotropic activity.

Differential roles of dihydropyridine calcium antagonist nifedipine, nitrendipine and amlodipine on gentamicin-induced renal tubular toxicity in rats

In the present study, we investigated the antioxidative potencies of dihydropyridine calcium antagonists prototype nifedipine, the second generation drug nitrendipine, and the long acting, third generation drug amlodipine on gentamicin-induced renal tubular toxicity in Sprague–Dawley rats. In addition, we analyzed the relationship between renal tubular cell apoptosis and the antioxidative properties of these dihydropyridine calcium antagonists. Results showed that treatment with gentamicin alone caused significant changes in the levels of urinary protein, urinary N-acetyl-beta- d-glucosaminidase, serum creatinine, and blood urea nitrogen. Nifedipine and amlodipine effectively reversed the effect of gentamicin on these parameters. In contrast, nitrendipine either had no effect or worsened gentamicin-induced changes in the levels of urinary protein, urinary N-acetyl-beta- d-glucosaminidase, serum creatinine, and blood urea nitrogen. Furthermore, gentamicin treatment caused significant increases in the levels of malondialdehyde, nitric oxide, nitric oxide synthase and significant decreases in the levels of reduced glutathione, glutathione-S-transferase, and superoxide dismutase in kidney tissues. These effects were dramatically reduced by nifedipine and amlodipine but not affected by nitrendipine. In addition to the biochemical changes, histopathological studies showed that gentamicin caused structural damages in the kidneys; renal tubular cell apoptosis, a decrease in Bcl-2 expression and an increase in Bax expression were observed in all rats treated with gentamicin, nifedipine and amlodipine effectively reversed the effect of gentamicin while nitrendipine worsened them. In conclusion, this study clearly indicated that nifedipine and amlodipine protected against gentamicin-induced nephrotoxicity while nitrendipine had little effect, or even worsened.

Inhibition of the Cardiac L-Type Calcium Channel Current by Antidepressant Drugs

Antidepressants inhibit many membrane receptors and ionic channels, including the L-type calcium channel. Here, we investigated the inhibition of calcium current (I(Ca)) by antidepressants in enzymatically isolated rat ventricular myocytes using whole-cell patch clamp. The molecular mechanism of inhibition was studied by comparing the voltage and state dependence of antidepressant inhibition of I(Ca) to the respective properties of calcium antagonists, and by studying the effect of (+/-)-1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl]phenyl)-3-pyridine carboxylic acid methyl ester (Bay K8644) or diltiazem on the inhibitory potency of the antidepressants. All selected antidepressants inhibited calcium currents reversibly and concentration-dependently. At a stimulation frequency of 0.33 Hz, the antidepressants imipramine, clomipramine, desipramine, amitriptyline, maprotiline, citalopram, and dibenzepin blocked I(Ca), with IC(50) values of 8.3, 11.6, 11.7, 23.2, 31.0, 64.5, and 364 muM. The antidepressant drugs shifted steady-state inactivation curves of I(Ca) to negative voltages. The extent of the shift was similar to that induced by diltiazem or verapamil, but it was significantly smaller than that induced by felodipine. The use-dependent component of the antidepressant-induced block was similar to that of diltiazem, and it was significantly more and less, respectively, than those of felodipine and verapamil. In the presence of Bay K8644, antidepressants were more effective in inhibiting I(Ca). However, the inhibitory effect of antidepressants was also augmented by diltiazem, suggesting that these drugs do not compete with diltiazem for a single binding site. These data suggest that antidepressants exert their inhibitory action on cardiac L-type calcium channels by a specific interaction at a receptor site similar to, but distinct from, the benzothiazepine site.

Bay w 9798, a Dihydropyridine Structurally Related to Nifedipine with No Calcium Channel-blocking Properties, Inhibits Tumour Necrosis Factor-α-induced Vascular Cell Adhesion Molecule-1 Expression in Endothelial Cells by Suppressing Reactive Oxygen Species Generation

Dihydropyridine-based calcium antagonists (DHPs) are widely used to treat hypertension. We have previously shown that nifedipine, one of the most popular DHPs, blocks tumour necrosis factor-alpha (TNF-alpha)-induced monocyte chemoattractant protein-1 as well as vascular cell adhesion molecule-1 (VCAM-1) expression in endothelial cells by suppressing reactive oxygen species generation (ROS). The molecular mechanism is still to be elucidated, however, because endothelial cells do not possess voltage-operated L-type calcium channels. The aim of this study was to determine in TNF-alpha-exposed human umbilical vein endothelial cells (HUVECs) whether and how Bay w 9798, a dihydropyridine structurally related to nifedipine with no calcium antagonistic properties, may suppress VCAM-1 expression, a key molecule which mediates the adhesion of monocytes to vasculature in the early stages of atherosclerosis. In HUVECs, 10 ng/ml TNF-alpha for 4 h stimulated ROS generation and subsequently upregulated VCAM-1 mRNA levels, both of which were dose-dependently blocked by Bay w 9798. The results demonstrated that Bay w 9798 inhibited VCAM-1 expression in TNF-alpha-exposed cells by suppressing ROS generation. They suggest that the anti-inflammatory and anti-oxidative properties of nifedipine and Bay w 9798 may be ascribed to the dihydropyridine structure, which is common to both molecules and has no calcium antagonistic ability.

Vasorelaxant effect of euxanthone in the rat thoracic aorta

This study was undertaken to investigate the effect of euxanthone on isolated rat thoracic aorta. Euxanthone concentration-dependently relaxed high K +-induced sustained contractions with IC 50 values of 32.28 ± 1.73 μM and this inhibition was antagonized by increasing the Ca 2+ concentration in the medium. These results indicated that euxanthone may have calcium antagonistic property. Euxanthone also relaxed norepinephrine (NE)-induced sustained contractions with IC 50 values of 32.50 ± 2.15 μM and this relaxant effect was unaffected by the removal of endothelium or by the presence of propranolol, indomethacin, glibenclamide or N ω-nitro- l-arginine. Moreover, euxanthone inhibited both the phasic and tonic contractions induced by NE in a concentration-dependent manner and showed more potent inhibition on phasic contraction ( P < 0.01). Pre-treatment with euxanthone inhibited vascular contraction induced by phorbol 12, 13-dibutyrate (PDBu), a protein kinase C (PKC) agonist, in either the presence or absence of Ca 2+ in the solution with IC 50 values of 20.15 ± 1.56 and 18.30 ± 1.62 μM, respectively. However, when the tissues were treated with euxanthone after the PDBu-induced contraction had reached a steady state, the tension was not affected by euxanthone. This study also showed that the inhibitory effect of pre-treatment of euxanthone was more potent than the post-treatment after the tension had reached a steady state. These results suggested that the vasorelaxation of euxanthone may be through multiple pathways involved PKC-mediated signal pathway and calcium-independent pathway besides the direct inhibition of calcium influx and its vasorelaxant effect is more active on calcium-independent pathway and more sensitive to the initial stage of contraction.

Withanolides, a new class of natural cholinesterase inhibitors with calcium antagonistic properties

The withanolides 1– 3 and 4– 5 isolated from Ajuga bracteosa and Withania somnifera, respectively, inhibited acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8) enzymes in a concentration-dependent fashion with IC 50 values ranging between 20.5 and 49.2 μM and 29.0 and 85.2 μM for AChE and BChE, respectively. Lineweaver–Burk as well as Dixon plots and their secondary replots indicated that compounds 1, 3, and 5 are the linear mixed-type inhibitors of AChE, while 2 and 4 are non-competitive inhibitors of AChE with K i values ranging between 20.0 and 45.0 μM. All compounds were found to be non-competitive inhibitors of BChE with K i values ranging between 27.7 and 90.6 μM. Molecular docking study revealed that all the ligands are completely buried inside the aromatic gorge of AChE, while compounds 1, 3, and 5 extend up to the catalytic triad. A comparison of the docking results showed that all ligands generally adopt the same binding mode and lie parallel to the surface of the gorge. The superposition of the docked structures demonstrated that the non-flexible skeleton of the ligands always penetrates the aromatic gorge through the six-membered ring A, allowing their simultaneous interaction with more than one subsite of the active center. The affinity of ligands with AChE was found to be the cumulative effects of number of hydrophobic contacts and hydrogen bonding. Furthermore, all compounds also displayed dose-dependent (0.005–1.0 mg/mL) spasmolytic and Ca 2+ antagonistic potentials in isolated rabbit jejunum preparations, compound 4 being the most active with an ED 50 value of 0.09 ± 0.001 mg/mL and 0.22 ± 0.01 μg/mL on spontaneous and K +-induced contractions, respectively. The cholinesterase inhibitory potential along with calcium antagonistic ability and safe profile in human neutrophil viability assay could make compounds 1– 5 possible drug candidates for further study to treat Alzheimer’s disease and associated problems.

Effect of estradiol 17β upon coronary artery vasoconstrictor response to methylergometrine maleate in female menopausal patients

Background Estrogens produce several beneficial effects upon the cardiovascular system. Amongst these, an endothelium-independent effect has been convincingly demonstrated only in vitro, while there is no evidence for such an effect in vivo. The aim of the present study was to evaluate the effect of acute administration of estradiol 17β upon coronary artery reactivity to methylergometrine in 16 menopausal patients with coronary artery disease. Methods Sixteen menopausal patients underwent coronary angiography at rest and after incremental doses of methylergometrine (intracoronary 2, 10, 30 μg) before and 20 min after either intracoronary estradiol 17β (20 ng/mL at 1 mL/min for 20 min; 8 patients) or placebo (Dextrose 5%, 1 mL/min; 8 patients). Results and conclusions No significant differences were observed in baseline coronary artery diameter or area between the 2 groups. No significant differences in the degree of coronary artery constriction were observed after either estradiol 17β or placebo at submaximal doses of methylergometrine. However, the degree of coronary artery constriction after maximal doses of methylergometrine was significantly attenuated by estradiol 17β compared to placebo (change in diameter: − 0.9 ± 4.5% vs. − 19 ± 6%, p < 0.001; change in area: − 3.2 ± 9% vs. − 32.2 ± 10%, p < 0.001). Estradiol 17β reduces coronary artery constriction following methylergometrine administration in menopausal patients with coronary artery disease. This effect may be related to the calcium-antagonist properties of the ovarian hormone. Condensed Abstract The effect of acute administration of estradiol 17β upon coronary artery reactivity to methylergometrine was evaluated by coronary angiography in 16menopausal women with CAD. The investigation was performed at rest and after incremental dose of methylergometrine before and 20 min after either intracoronary estradiol 17β or placebo. Significant differences were not observed either in the baseline coronary artery diameter or the area between the 2groups, or in the degree of coronary artery constriction after either estradiol 17β or placebo at submaximal doses of methylergometrine. Coronary artery constriction after maximal doses of methylergometrine resulted attenuated by estradiol 17β for its calcium-antagonist properties.