Endothelium-independent vasorelaxant effect of sodium ferulate on rat thoracic aorta

AimsThis study was designed to investigate the effects of sodium ferulate (SF) on rat isolated thoracic aortas and the possible mechanisms.Main methodsIsometric tension was recorded in response to drugs in organ bath. Cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) was measured using Fluo-3 in cultured rat aortic smooth muscle cells (RASMC).Key findingsSF (0.1–30 mM) relaxed the isolated aortic rings precontracted with phenylephrine (PE) and high-K⁺ in a concentration-dependent manner with respective pD₂ of 2.7 ± 0.02 and 2.6 ± 0.06. Mechanical removal of endothelium did not significantly modify the SF-induced relaxation. In Ca²⁺-free solution, SF noticeably inhibited extracellular Ca²⁺-induced contraction in high-K⁺ and PE pre-challenged rings, and suppressed the transient contraction induced by PE and caffeine. The vasorelaxant effect of SF was unaffected by various K⁺ channel blockers such as tetraethylammonium, glibenclamide, 4-aminopyridine, and barium chloride. In addition, SF concentration-dependently reduced the contraction induced by phorbol-12-myristate-13-acetate, an activator of protein kinase C (PKC), in the absence of extracellular Ca²⁺, with the pD₂ of 2.9 ± 0.03. In RASMC, SF had no effect on PE- or KCl-induced [Ca²⁺]_i increase either in the presence or in the absence of external Ca²⁺.SignificanceThese results indicate that SF acts directly as a non-selective relaxant to vascular smooth muscle. The direct inhibition of the common pathway after [Ca²⁺]_i increase may account for the SF-induced relaxation in Ca²⁺-dependent contraction, while the blockage of the PKC-mediated contractile mechanism is likely responsible for the SF-induced relaxation in Ca²⁺-independent contraction.     

KMUP-1 ameliorates monocrotaline-induced pulmonary arterial hypertension through the modulation of Ca2+ sensitization and K+-channel

AimsThis study investigates the actions of KMUP-1 on RhoA/Rho-kinase (ROCK)-dependent Ca²⁺ sensitization and the K⁺-channel in chronic pulmonary arterial hypertension (PAH) rats.Main methodsSprague–Dawley rats were divided into control, monocrotaline (MCT), and MCT + KMUP-1 groups. PAH was induced by a single intraperitoneal injection (i.p.) of MCT (60 mg/kg). KMUP-1 (5 mg/kg, i.p.) was administered once daily for 21 days to prevent MCT-induced PAH. All rats were sacrificed on day 22.Key findingsMCT-induced increased right ventricular systolic pressure (RVSP) and right ventricular hypertrophy were prevented by KMUP-1. In myograph experiments, KCl (80 mM), phenylephrine (10 µM) and K⁺ channel inhibitors (TEA, 10 mM; paxilline, 10 µM; 4-AP, 5 mM) induced weak PA contractions in MCT-treated rats compared to controls, but the PA reactivity was restored in MCT + KMUP-1-treated rats. By contrast, in β-escin- or α-toxin-permeabilized PAs, CaCl₂-induced (1.25 mM, pCa 5.1) contractions were stronger in MCT-treated rats, and this action was suppressed in MCT + KMUP-1-treated rats. PA relaxation in response to the ROCK inhibitor Y27632 (0.1 μM) was much higher in MCT-treated rats than in control rats. In Western blot analysis, the expression of Ca²⁺-activated K⁺ (BK_Ca) and voltage-gated K⁺ channels (Kv2.1 and Kv1.5), and ROCK II proteins was elevated in MCT-treated rats and suppressed in MCT + KMUP-1-treated rats. We suggest that MCT-treated rats upregulate K⁺-channel proteins to adapt to chronic PAH.SignificanceKMUP-1 protects against PAH and restores PA vessel tone in MCT-treated rats, attributed to alteration of Ca²⁺ sensitivity and K⁺-channel function.     

Vasoconstriction triggered by hydrogen sulfide: Evidence for Na+,K+,2Cl-cotransport and L-type Ca2+ channel-mediated pathway

ObjectivesThis study examined the dose-dependent actions of hydrogen sulfide donor sodium hydrosulphide (NaHS) on isometric contractions and ion transport in rat aorta smooth muscle cells (SMC).MethodsIsometric contraction was measured in ring aortas segments from male Wistar rats. Activity of Na⁺/K⁺-pump and Na⁺,K⁺,2Cl^-cotransport was measured in cultured endothelial and smooth muscle cells from the rat aorta as ouabain-sensitive and ouabain-resistant, bumetanide-sensitive components of the ⁸⁶Rb influx, respectively.ResultsNaHS exhibited the bimodal action on contractions triggered by modest depolarization ([K⁺]_o=30 mM). At 10^?4 M, NaHS augmented contractions of intact and endothelium-denuded strips by ~ 15% and 25%, respectively, whereas at concentration of 10^?3 M it decreased contractile responses by more than two-fold. Contractions evoked by 10^?4 M NaHS were completely abolished by bumetanide, a potent inhibitor of Na⁺,K⁺,2Cl^-cotransport, whereas the inhibition seen at 10^?3 M NaHS was suppressed in the presence of K⁺ channel blocker TEA. In cultured SMC, 5×10^?5 M NaHS increased Na⁺,K⁺,2Cl^- - cotransport without any effect on the activity of this carrier in endothelial cells. In depolarized SMC, ⁴⁵Ca influx was enhanced in the presence of 10^?4 M NaHS and suppressed under elevation of [NaHS] up to 10^?3 M. ⁴⁵Ca influx triggered by 10^?4 M NaHS was abolished by bumetanide and L-type Ca²⁺ channel blocker nicardipine.ConclusionsOur results strongly suggest that contractions of rat aortic rings triggered by low doses of NaHS are mediated by activation of Na⁺,K⁺,2Cl^-cotransport and Ca²⁺ influx via L-type channels.     

Magnolol inhibits colonic motility through down-regulation of voltage-sensitive L-type Ca2+ channels of colonic smooth muscle cells in rats

This study aimed to investigate the effect of magnolol (5,5′-diallyl-2,2′-biphenyldiol) on contraction in distal colonic segments of rats and the underlying mechanisms. Colonic segments were mounted in organ baths for isometric force measurement. Whole-cell voltage-sensitive L-type Ca²⁺ currents were recorded on isolated single colonic smooth muscle cells using patch-clamp technique. The spontaneous contractions and acetylcholine (ACh)- and Bay K 8644-induced contractions were inhibited by magnolol (3–100 μM). In the presence of Bay K8644 (100 nM), magnolol (10–100 μM) inhibited the contraction induced by 10 μM ACh. By contrast, tetrodotoxin (100 nM) and N_ώ-nitro-l-arginine methyl ester (l-NAME 100 μM) did not change the inhibitory effect of magnolol (10 μM). In addition, magnolol (3–100 μM) inhibited the L-type Ca²⁺ currents. The present results suggest that magnolol inhibits colonic smooth muscle contraction through downregulating L-type Ca²⁺ channel activity.     

Involvement of plasma membrane Ca2+ channels,IP3 receptors,and ryanodine receptors in the generation of spontaneous rhythmic contractions of the cricket lateral oviduct

In the present study, the isolated cricket (Gryllus bimaculatus) lateral oviduct exhibited spontaneous rhythmic contractions (SRCs) with a frequency of 0.29 ± 0.009 Hz (n = 43) and an amplitude of 14.6 ± 1.25 mg (n = 29). SRCs completely disappeared following removal of extracellular Ca²⁺ using a solution containing 5 mM EGTA. Application of the non-specific Ca²⁺ channel blockers Co²⁺, Ni²⁺, and Cd²⁺ also decreased both the frequency and amplitude of SRCs in dose-dependent manners, suggesting that Ca²⁺ entry through plasma membrane Ca²⁺ channels is essential for the generation of SRCs. Application of ryanodine (30 μM), which depletes intracellular Ca²⁺ by locking ryanodine receptor (RyR)-Ca²⁺ channels in an open state, gradually reduced the frequency and amplitude of SRCs. A RyR antagonist, tetracaine, reduced both the frequency and amplitude of SRCs, whereas a RyR activator, caffeine, increased the frequency of SRCs with a subsequent increase in basal tonus, indicating that RyRs are essential for generating SRCs. To further investigate the involvement of phospholipase C (PLC) and inositol 1,4,5-trisphosphate receptors (IP₃Rs) in SRCs, we examined the effect of a PLC inhibitor, U73122, and an IP₃R antagonist, 2-aminoethoxydiphenyl borate (2-APB), on SRCs. Separately, U73122 (10 μM) and 2-APB (30–50 μM) both significantly reduced the amplitude of SRCs with little effect on their frequency, further indicating that the PLC/IP₃R signaling pathway is fundamental to the modulation of the amplitude of SRCs. A hypotonic-induced increase in the frequency and amplitude of SRCs and a hypertonic-induced decrease in the frequency and amplitude of SRCs indicated that mechanical stretch of the lateral oviduct is involved in the generation of SRCs. The sarcoplasmic reticulum Ca²⁺-pump ATPase inhibitors thapsigargin and cyclopiazonic acid impaired or suppressed the relaxation phase of SRCs. Taken together, the present results indicate that Ca²⁺ influx through plasma membrane Ca²⁺ channels and Ca²⁺ release from RyRs play an essential role in pacing SRCs and that Ca²⁺ release from IP₃Rs may play a role in modulating the amplitude of SRCs, probably via activation of PLC.     

Synthesis and biological evaluation of 18F labeled fluoro-oligo-ethoxylated 4-benzylpiperazine derivatives for sigma-1 receptor imaging

We report the synthesis and evaluation of a series of fluoro-oligo-ethoxylated 4-benzylpiperazine derivatives as potential σ₁ receptor ligands. In vitro competition binding assays showed that 1-(1,3-benzodioxol-5-ylmethyl)-4-(4-(2-fluoroethoxy)benzyl)piperazine (6) exhibits low nanomolar affinity for σ₁ receptors (K_i = 1.85 ± 1.59 nM) and high subtype selectivity (σ₂ receptor: K_i = 291 ± 111 nM; K_iσ₂/K_iσ₁ = 157). [¹⁸F]6 was prepared in 30–50% isolated radiochemical yield, with radiochemical purity of >99% by HPLC analysis after purification, via nucleophilic ¹⁸F^? substitution of the corresponding tosylate precursor. The log D_{pH 7.4} value of [¹⁸F]6 was found to be 2.57 ± 0.10, which is within the range expected to give high brain uptake. Biodistribution studies in mice demonstrated relatively high concentration of radiotracers in organs known to contain σ₁ receptors, including the brain, lungs, kidneys, heart, and spleen. Administration of haloperidol 5 min prior to injection of [¹⁸F]6 significantly reduced the concentration of radiotracers in the above-mentioned organs. The accumulation of radiotracers in the bone was quite low suggesting that [¹⁸F]6 is relatively stable to in vivo defluorination. The ex vivo autoradiography in rat brain showed high accumulation of radiotracers in the brain areas known to possess high expression of σ₁ receptors. These findings suggest that [¹⁸F]6 is a suitable radiotracer for imaging σ₁ receptors with PET in vivo.     

Regionally diverse mitochondrial calcium signaling regulates spontaneous pacing in developing cardiomyocytes

The quintessential property of developing cardiomyocytes is their ability to beat spontaneously. The mechanisms underlying spontaneous beating in developing cardiomyocytes are thought to resemble those of adult heart, but have not been directly tested. Contributions of sarcoplasmic and mitochondrial Ca²⁺-signaling vs. I_f-channel in initiating spontaneous beating were tested in human induced Pluripotent Stem cell-derived cardiomyocytes (hiPS-CM) and rat Neonatal cardiomyocytes (rN-CM). Whole-cell and perforated-patch voltage-clamping and 2-D confocal imaging showed: (1) both cell types beat spontaneously (60–140/min, at 24 °C); (2) holding potentials between −70 and 0 mV had no significant effects on spontaneous pacing, but suppressed action potential formation; (3) spontaneous pacing at −50 mV activated cytosolic Ca²⁺-transients, accompanied by in-phase inward current oscillations that were suppressed by Na⁺-Ca²⁺-exchanger (NCX)- and ryanodine receptor (RyR2)-blockers, but not by Ca²⁺- and I_f-channels blockers; (4) spreading fluorescence images of cytosolic Ca²⁺-transients emanated repeatedly from preferred central cellular locations during spontaneous beating; (5) mitochondrial un-coupler, FCCP at non-depolarizing concentrations (∼50 nM), reversibly suppressed spontaneous pacing; (6) genetically encoded mitochondrial Ca²⁺-biosensor (mitycam-E31Q) detected regionally diverse, and FCCP-sensitive mitochondrial Ca²⁺-uptake and release signals activating during I_NCX oscillations; (7) I_f-channel was absent in rN-CM, but activated only negative to −80 mV in hiPS-CM; nevertheless blockers of I_f-channel failed to alter spontaneous pacing.     

Synthesis,radiolabeling and preliminary in vivo evaluation of [18F]FE-PE2I,a new probe for the dopamine transporter

A new dopamine transporter (DAT) ligand, (E)-N-(3-iodoprop-2-enyl)-2β-carbofluoroethoxy-3β-(4′-methyl-phenyl) nortropane (FE-PE2I, 6), derived from PE2I (1), was prepared and found to be a potent inhibitor of rodent DAT in vitro. Compound 6 was radiolabelled with fluorine-18 (t_1/2 = 109.8 min) for PET studies in monkeys. In vivo PET measurements showed a regional distribution in brain that corresponds to the known distribution of DAT. This binding was specific, reversible and the kinetics of [¹⁸F]6 binding in brain were faster than for its lead compound, [¹¹C]1. The possible presence of a hydroxymethyl-radiometabolite formed by oxidation in the 3β-benzylic position of [¹⁸F]6 warrants further detailed evaluation of the metabolism of [¹⁸F]6. [¹⁸F]6 is a potential radioligand for imaging DATs in the human brain with PET.     

Synthesis,spectral and magnetical characterization of monomeric [Cu(2-NO2bz)2(3-pyme)2(H2O)2] and polymeric [Cu{3,5-(NO2)2bz}2(3-pyme)2]n

Two new complexes of composition [Cu(2-NO₂bz)₂(3-pyme)₂(H₂O)₂] (1) and/or [Cu{3,5-(NO₂)₂bz}₂(3-pyme)₂] (2) (3-pyme = 3-pyridylmethanol, ronicol or 3-pyridylcarbinol, 2-NO₂bz = 2-nitrobenzoate and 3,5-(NO₂)₂bz = 3,5-dinitrobenzoate) have been prepared and studied by elemental analysis, electronic, infrared and EPR spectroscopy, magnetic susceptibility measurements and the structure of both complexes has been solved. Complex (1) shows an unusual molecular type of structure consisting of the [Cu(2-NO₂bz)₂(3-pyme)₂(H₂O)₂] molecules held together by hydrogen bonds and van der Waals interactions. Complex (2) exhibits a polymeric chain-like structure [Cu{3,5-(NO₂)₂bz}₂(3-pyme)₂]_n with copper atoms doubly bridged by two 3-pyridylmethanol molecules and the polymeric molecules are held together by van der Waals interactions. Complex (1) exhibits a magnetic moment μ_eff = 1.84 B.M. at 300 K that remains nearly constant within the temperature region (5–300 K). Further cooling results in lowering the magnetic moment to μ_eff = 1.82 B.M. at 1.8 K. The magnetic susceptibility temperature dependence obeys Curie–Weiss law with Curie constant of 0.423 cm³ K mol⁻¹ and with Weiss constant of −0.06 K. The magnetic moment of (2) exhibits a small increase with a decrease in the temperature (μ_eff = 1.80 B.M. at 300 K and μ_eff = 1.85 B.M. at 1.8 K) with Curie constant of 0.409 cm³ K mol⁻¹ and with Weiss constant of +1.1 K, which can indicate a very weak ferromagnetic interaction between the copper atoms within the chain. Applying the molecular field model resulted in obtaining zJ′ values −0.08 cm⁻¹ for complex (1), and −0.07 cm⁻¹ for complex (2), respectively, that could characterize intermolecular and interchain interactions transmitted through π–π stacking.     

New N-substituted 9-azabicyclo[3.3.1]nonan-3α-yl phenylcarbamate analogs as σ2 receptor ligands: Synthesis,in vitro characterization,and evaluation as PET imaging and chemosensitization agents

A series of N-substituted 9-azabicyclo[3.3.1]nonan-3α-yl phenylcarbamate analogs were synthesized. Among them, WC-26 and WC-59 were identified as the most potent σ₂ receptor ligands (K_i = 2.58 and 0.82 nM, respectively) with high selectivity against σ₁ (K_i of σ₁/σ₂ ratio = 557 and 2087, respectively). [¹⁸F]WC-59 was radiolabeled via a nucleophilic substitution of a mesylate precursor by [¹⁸F]fluoride, and in vitro direct binding studies of [¹⁸F]WC-59 were conducted using membrane preparations from murine EMT-6 solid breast tumors. The results indicate that [¹⁸F]WC-59 binds specifically to σ₂ receptors in vitro (K_d = ～2 nM). Biodistribution studies of [¹⁸F]WC-59 in EMT-6 tumor-bearing mice indicated that the tracer was a less suitable candidate for clinical imaging studies than existing F-18 labeled σ₂ receptor ligands. The ability of WC-26 to enhance the cytotoxic effects of the chemotherapy drug, doxorubicin, was evaluated in cell culture using the mouse breast tumor EMT-6 and the human tumor MDA-MB435. WC-26 greatly increased the ability of doxorubicin to kill these two tumor cell lines in vitro. These results indicate that WC-26 is potentially a useful chemosensitizer for the treatment of cancer when combined with conventional chemotherapeutics.     

Synthesis and in vivo evaluation of [18F]2-(4-(4-(2-(2-fluoroethoxy)phenyl)piperazin-1-yl)butyl)-4-methyl-1,2,4-triazine-3,5(2H,4H)-dione ([18F]FECUMI-101) as an imaging probe for 5-HT1A receptor agonist in nonhuman primates

The 5-HT_1AR partial agonist PET radiotracer, [¹¹C]CUMI-101, has advantages over an antagonist radiotracer as it binds preferentially to the high affinity state of the receptor and thereby provides more functionally meaningful information. The major drawback of C-11 tracers is the lack of cyclotron facility in many health care centers thereby limiting widespread clinical or research use. We identified the fluoroethyl derivative, 2-(4-(4-(2-(2-fluoroethoxy)phenyl)piperazin-1-yl)butyl)-4-methyl-1,2,4-triazine-3,5(2H,4H)dione (FECUMI-101) (K_i = 0.1 nM; E_max = 77%; EC₅₀ = 0.65 nM) as a partial agonist 5-HT_1AR ligand of the parent ligand CUMI-101. FECUMI-101 is radiolabeled with F-18 by O-fluoroethylation of the corresponding desmethyl analogue (1) with [¹⁸F]fluoroethyltosylate in DMSO in the presence of 1.6 equiv of K₂CO₃ in 45 ± 5% yield (EOS). PET shows [¹⁸F]FECUMI-101 binds specifically to 5-HT_1AR enriched brain regions of baboon. The specificity of [¹⁸F]FECUMI-101 binding to 5-HT_1AR was confirmed by challenge studies with the known 5-HT_1AR ligand WAY100635. These findings indicate that [¹⁸F]FECUMI-101 can be a viable agonist ligand for the in vivo quantification of high affinity 5-HT_1AR with PET.     

Design,synthesis, functional and structural characterization of an inhibitor of N-acetylneuraminate-9-phosphate phosphatase: Observation of extensive dynamics in an enzyme/inhibitor complex

The design, synthesis and characterization of a phosphonate inhibitor of N-acetylneuraminate-9-phosphate phosphatase (HDHD4) is described. Compound 3, where the substrate C-9 oxygen was replaced with a nonlabile CH₂ group, inhibits HDHD4 with a binding affinity (IC₅₀ 11 μM) in the range of the native substrate Neu5Ac-9-P (compound 1, K_m 47 μM). Combined SAR, modeling and NMR studies are consistent with the phosphonate group in inhibitor 3 forming a stable complex with native Mg²⁺. In addition to this key interaction, the C-1 carboxylate of the sugar interacts with a cluster of basic residues, K141, R104 and R72. Comparative NMR studies of compounds 3 and 1 with Ca²⁺ and Mg²⁺ are indicative of a highly dynamic process in the active site for the HDHD4/Mg²⁺/3 complex. Possible explanations for this observation are discussed.     

Synthesis and monoamine transporter affinity of 3α-arylmethoxy-3β-arylnortropanes

A series of 3-arylnortrop-2-enes and 3α-arylmethoxy-3β-arylnortropanes were synthesized and evaluated for binding affinity at monoamine transporters. The 3-(3,4-dichlorophenyl)nortrop-2-ene (6e) exhibited high affinity for the SERT (K_i = 0.3 nM). The 3α-arylmethoxy-3β-arylnortropanes were generally SERT selective with the 3α-(3.4-dichlorophenylmethoxy)-3βphenylnortrop-2-ene (7c) possessing subnanomolar potency (K_i = 0.061 nM). However, 3α-(3,4-dichlorophenylmethoxy)-3β-phenylnortrop-2-ene (7b) exhibited high affinity at all three transporters [(DAT K_i = 22 nM), (SERT K_i = 6 nM) and (NET K_i = 101 nM)].     

Pyrrolidine analogs of GZ-793A: Synthesis and evaluation as inhibitors of the vesicular monoamine transporter-2 (VMAT2)

Central heterocyclic ring size reduction from piperidinyl to pyrrolidinyl in the vesicular monoamine transporter-2 (VMAT2) inhibitor GZ-793A and its analogs resulted in novel N-propane-1,2(R)-diol analogs 11a–i. These compounds were evaluated for their affinity for the dihydrotetrabenazine (DTBZ) binding site on VMAT2 and for their ability to inhibit vesicular dopamine (DA) uptake. The 4-difluoromethoxyphenethyl analog 11f was the most potent inhibitor of [³H]-DTBZ binding (K_i = 560 nM), with 15-fold greater affinity for this site than GZ-793A (K_i = 8.29 μM). Analog 11f also showed similar potency of inhibition of [³H]-DA uptake into vesicles (K_i = 45 nM) compared to that for GZ-793A (K_i = 29 nM). Thus, 11f represents a new water-soluble inhibitor of VMAT function.     

Evaluation of (Z)-2-((1-benzyl-1H-indol-3-yl)methylene)-quinuclidin-3-one analogues as novel,high affinity ligands for CB1 and CB2 cannabinoid receptors

A small library of N-benzyl indolequinuclidinone (IQD) analogs has been identified as a novel class of cannabinoid ligands. The affinity and selectivity of these IQDs for the two established cannabinoid receptor subtypes, CB1 and CB2, was evaluated. Compounds 8 (R = R² = H, R¹ = F) and 13 (R = COOCH₃, R¹ = R² = H) exhibited high affinity for CB2 receptors with K_i values of 1.33 and 2.50 nM, respectively, and had lower affinities for the CB1 receptor (K_i values of 9.23 and 85.7 nM, respectively). Compound 13 had the highest selectivity of all the compounds examined, and represents a potent cannabinoid ligand with 34-times greater selectivity for CB2R over CB1R. These findings are significant for future drug development, given recent reports demonstrating beneficial use of cannabinoid ligands in a wide variety of human disease states including drug abuse, depression, schizophrenia, inflammation, chronic pain, obesity, osteoporosis and cancer.     

Influence of ionic interactions on essential oil and phenolic diterpene composition of Dalmatian sage (Salvia officinalis L.)

The potential of four essential cations (K⁺, Ca²⁺, Mg²⁺ and Fe²⁺) to alleviate salt toxicity was studied in sage (Salvia officinalis L.) plants grown in pots. Two concentrations of the following chloride salts: KCl, CaCl₂, MgCl₂ and FeCl_3, were used together with 100 mM NaCl to study the effects of these nutrients on plant growth, leaf essential oils (EOs) and phenolic diterpenes composition. The sage plants accumulated Na⁺ in their leaves (includers); this has affected secondary metabolites’ biosynthesis. Treatment with 100 mM NaCl slightly decreased borneol and viridiflorol, while increased manool concentrations. Addition of KCl, CaCl₂ and MgCl₂ increased considerably in a dose-dependent manner the oxygen-containing monoterpenes (1.8-cineole, camphor, β-thujone and borneol) in 100 mM NaCl-treated sage. Whereas, the contents of viridiflorol decreased further with the addition of KCl in 100 mM NaCl-treated sage. Our results suggest that the changes in EOs composition were more related to K⁺ and Ca²⁺ availability than to Na⁺ toxicity. Furthermore, treatment with NaCl decreased by 50% carnosic acid (CA), a potent antioxidant, content in the leaves. K⁺ and Ca²⁺ promoted the accumulation of CA and its methoxylated form (MCA) in the leaves. The concentration of CA was positively correlated with leaf K⁺ (r = 0.56, P = 0.01) and Ca²⁺ (r = 0.44, P = 0.05) contents. It appears that different salt applications in combination with NaCl treatments had a profound effect on EOs and phenolic diterpene composition in sage. Therefore, ionic interactions may be carefully considered in the cultivation of this species to get the desired concentrations of these secondary metabolites in leaf extracts.     

Synthesis,crystal structure and magnetic properties of the spin crossover system [Fe(pq)3]2+

Three new compounds formulated (ClO₄)₂[Fe(pq)₃] (1), (BF₄)₂[Fe(pq)₃] · EtOH (2) and {(ClO₄)[MnCr(C₂O₄)₃][Fe(pq)₂(H₂O)₂]} (3), where pq is 2,2′-pyridylquinoline, have been synthesised and characterised. Despite the different crystal packing exhibited by 1 and 2, the cationic species [Fe(pq)₃]²⁺ are structurally quite similar. At 293 K, the Fe–N bond lengths are characteristic of the iron(II) in the high-spin state. In contrast to 1, 2 undergoes a continuous spin transition. Indeed, at 95 K its structure experiences a noticeable change in the Fe–N bonds and angles, i.e. the Fe–N bonds shorten by 0.194 Å on the average. The magnetic behaviour confirms that 1 is fully high-spin in the 4–300 K temperature range while 2 shows a spin transition centred at T_1/2 = 150 K. The corresponding enthalpy, entropy and interaction parameter are ΔH = 7.49 kJ mol^?1, ΔS = 50 J K^?1 mol^?1and Γ = 1.35 kJ mol^?1. Compound 3 has been obtained as a microcrystalline powder. The magnetic properties of 3 point at the occurrence of ferromagnetic coupling below 100 K and the onset of a ferromagnetic ordering below 10 K (Weiss constant equal to 6.8 K). The Mössbauer spectra of 3 show the occurrence of a magnetic order at T ? 4.2 K.     

Evaluation of the association of mercury(II) with some dicysteinyl tripeptides

The present study was undertaken to gain insight into the associations of mercury(II) with dicysteinyl tripeptides in buffered media at pH 7.4. We investigated the effects of increasing the distance between cysteinyl residues on mercury(II) associations and complex formations. The peptide–mercury(II) formation constants and their associated thermodynamic parameters in 3-(N-morpholino)propanesulfonic acid (MOPS) buffered solutions were evaluated by isothermal titration calorimetry. Complexes formed in different relative ratios of mercury(II) to cysteinyl peptides in ammonium formate buffered solutions were characterized by LTQ Orbitrap mass spectrometry. The results from these studies show that n-alkyl dicysteinyl peptides (CP 1–4), and an aryl dicysteinyl peptide (CP 5) can serve as effective “double anchors” to accommodate the coordination sites of mercury(II) to form predominantly one-to-one Hg(peptide) complexes. The aryl dicysteinyl peptide (CP 5) also forms the two-to-two Hg₂(peptide)₂ complex. In the presence of excess peptide, Hg(peptide)₂ complexes are also detected. Notably, increasing the distance between the ligating groups or “anchor points” in CP 1–5 does not significantly affect their affinity for mercury(II). However, the enthalpy change (ΔH) values (ΔH₁ ∼ −91 kJ mol⁻¹ and ΔH₂ ∼ −66 kJ mol⁻¹) for complex formation between CP 4 and 5 with mercury(II) are about one and a half times larger than the related values for CP 1, 2 and 3 (ΔH₁ ∼ −66 kJ mol⁻¹ and ΔH₂ ∼ 46 kJ mol⁻¹). The corresponding entropy change (ΔS) values (ΔS₁ ∼ −129 J K⁻¹ mol⁻¹ and ΔS₂ ∼ −116 J K⁻¹ mol⁻¹) of the structurally larger dicysteinyl peptides CP 4 and 5 are less entropically favorable than for CP 1, 2 and 3 (ΔS₁ ∼ −48 J K⁻¹ mol⁻¹ and ΔS₂ ∼ −44 J K⁻¹ mol⁻¹). Generally, these associations result in a decrease in entropy, indicating that these peptide–mercury complexes potentially form highly ordered structures. The results from this study show that dicysteinyl tripeptides are effective in binding mercury(II) and they are promising motifs for the design of multi-cysteinyl peptides for binding more than one mercury(II) ion per peptide.     

Synthesis and evaluation of novel azetidine analogs as potent inhibitors of vesicular [3H]dopamine uptake

Lobelane analogs that incorporate a central piperidine or pyrrolidine moiety have previously been reported by our group as potent inhibitors of VMAT2 function. Further central ring size reduction of the piperidine moiety in lobelane to a four-membered heterocyclic ring has been carried out in the current study to afford novel cis-and trans-azetidine analogs. These azetidine analogs (15a–15c and 22a–22c) potently inhibited [³H]dopamine (DA) uptake into isolated synaptic vesicles (K_i ? 66 nM). The cis-4-methoxy analog 22b was the most potent inhibitor (K_i = 24 nM), and was twofold more potent that either lobelane (2a, K_i = 45 nM) or norlobelane (2b, K_i = 43 nM). The trans-methylenedioxy analog, 15c (K_i = 31 nM), was equipotent with the cis-analog, 22b, in this assay. Thus, cis- and trans-azetidine analogs 22b and 15c represent potential leads in the discovery of new clinical candidates for the treatment of methamphetamine abuse.     

Total internal reflectance fluorescence imaging of genetically engineered ryanodine receptor-targeted Ca2+ probes in rat ventricular myocytes

The details of cardiac Ca²⁺ signaling within the dyadic junction remain unclear because of limitations in rapid spatial imaging techniques, and availability of Ca²⁺ probes localized to dyadic junctions. To critically monitor ryanodine receptors’ (RyR2) Ca²⁺ nano-domains, we combined the use of genetically engineered RyR2-targeted pericam probes, (FKBP-YCaMP, K_d = 150 nM, or FKBP-GCaMP6, K_d = 240 nM) with rapid total internal reflectance fluorescence (TIRF) microscopy (resolution, ∼80 nm). The punctate z-line patterns of FKBP,²-targeted probes overlapped those of RyR2 antibodies and sharply contrasted to the images of probes targeted to sarcoplasmic reticulum (SERCA2a/PLB), or cytosolic Fluo-4 images. FKBP-YCaMP signals were too small (∼20%) and too slow (2–3 s) to detect Ca²⁺ sparks, but the probe was effective in marking where Fluo-4 Ca²⁺ sparks developed. FKBP-GCaMP6, on the other hand, produced rapidly decaying Ca²⁺ signals that: a) had faster kinetics and activated synchronous with I_Ca³ but were of variable size at different z-lines and b) were accompanied by spatially confined spontaneous Ca²⁺ sparks, originating from a subset of eager sites. The frequency of spontaneously occurring sparks was lower in FKBP-GCaMP6 infected myocytes as compared to Fluo-4 dialyzed myocytes, but isoproterenol enhanced their frequency more effectively than in Fluo-4 dialyzed cells. Nevertheless, isoproterenol failed to dissociate FKBP-GCaMP6 from the z-lines. The data suggests that FKBP-GCaMP6 binds predominantly to junctional RyR2s and has sufficient on-rate efficiency as to monitor the released Ca²⁺ in individual dyadic clefts, and supports the idea that β-adrenergic agonists may modulate the stabilizing effects of native FKBP on RyR2.