Methyl- and propylacetamidinates of lanthanides: Structures, catalytic and some physical properties

The acetamidinates {[MeNC(Me)NMe]₂Ln}₂[μ-η²-η²-MeNC(Me)NMe]₂ (Ln = Y (1), Dy (2)) and {[PrⁿNC(Me)NPrⁿ]₂Y}₂[μ-η²-η²-PrⁿNC(Me)NPrⁿ]₂ (3) have been prepared by the reactions of amides Ln[N(SiMe₃)₂]₃ with respective N,N′-disubstituted amidines MeNC(Me)NHMe or PrⁿNC(Me)NHPrⁿ. The reaction of Er[N(SiMe₃)₂]₃ with excess of monosubstituted amidine HNC(Me)NHPrⁱ or in a ratio of 1:2 resulted in the formation of compound {Er[NC(Me)NHPrⁱ]₃}_x (4). The same reaction with 1:1 ratio yielded heteroleptic complex {Er[N(SiMe₃)₂]₂[NC(Me)NHPrⁱ]}_x (5). The complexes 1, 2 and 3 have similar structures and contain four terminal and two μ-η²:η²-N,N-bridging amidinate groups binding the metal atoms. Volatility of 1, 2 and 3 is comparable to that of known monomeric La[PrⁱNC(R)NPrⁱ]₃. Compound 1 efficiently catalyzes the ring-opening polymerization of rac-lactide to give polylactide with M_n 53 085 and polydispersity 1.84.     

Synthesis and characterisation of the halogenated azanonaboranes [(PrNH2)B8H10XNHPr] (X=Cl, Br, I) and their unexpected hydrolysis to hypho-[B5H10(μ-NHPr)]

Treatment of [(ⁱPrNH₂)B₈H₁₁NHⁱPr] with elemental halogen affords the 8-exo-halogen-substituted derivatives [(ⁱPrNH₂)B₈H₁₀XNHⁱPr] (X=Cl, Br, I). The structures of all three compounds are confirmed by NMR spectroscopy and mass spectrometry, and (for X=Br) by an X-ray diffraction study. The bromoazanonaborane undergoes hydrolytic decomposition to the new five-vertex compound [B₅H₁₀(μ-NHⁱPr)] of hypho-type structure.     

Syntheses of 1-[2-(Phosphonomethoxy)Alkyl]Thymine Monophosphates and an Evaluation of their Inhibitory Activity Toward Human Thymidine Phosphorylase

A series of new monophosphates of 1-[2-(phosphonomethoxy)alkyl]thymines, such as PMPTp_, 3-MeO-PMPTp, HPMPTp, and FPMPTp, were synthesized and tested for their ability to inhibit human thymidine phosphorylase. Kinetic measurements of enzyme activity were performed using thymidine and inorganic phosphate as the substrates. The data show that some monophosphates provide a considerable increase of the multisubstrate inhibitory effect. The highest inhibitory potency was found with (R)-FPMPTp 4c (K _i ^dT = 4.09 ± 0.47 μM, K _i(P_i) = 2.13 ± 0.29 μM) and (R) 3-MeO-PMPTp 4d (K _i ^dT = 5.78 ± 0.71 μM, K _i(P_i) = 2.71 ± 0.37 μM).     

New natural cholinesterase inhibiting and calcium channel blocking quinoline alkaloids

During this study, one new coumarin; 7-O-β-D-glucopyranoside-2H-1-benzopyran-2-one (1) and three quinoline alkaloids; 3-hydroxy, 2, 2, 6-trimethyl–3, 4, 5, 6-tetrahydro-2H-pyrano[3,2-c] quinoline 5-one (2), ribalinine (3) and methyl isoplatydesmine (4) were isolated from the aerial parts of Skimmia laureola and their structures established by spectroscopic studies. Compounds 2-4 were found to be linear mixed type inhibitors of acetylcholinesterase (K_i = 110.0, 30.0 and 30.0 μM, respectively). Compounds 2 and 3 were also found to be linear mixed type inhibitors of butyrylcholinesterase, while compound 4 was a noncompetitive inhibitor of the enzyme (K_i = 90.0, 70.0 and 19.0 μM, respectively). The inhibition of acetyl- and butyryl-cholinesterase enzymes persists as the most promising therapeutic strategy for activating the impaired cholinergic functions in Alzheimer's disease and related dementias.

Compound 4 also showed dose-dependent spasmolytic activity in the isolated rabbit jejunum intestinal preparation by relaxing the spontaneous (EC₅₀ = 0.1 mg/mL) and K⁺-induced contractions (EC₅₀ = 0.4 mg/mL), suggesting that the spasmolytic effect of compound 4 is mediated through the blockade of voltage-dependent Ca^{2 +} channels.     

Preparation and physico-chemical studies of some chloride and alkoxide isopropoxymetallates of cobalt(II) containing {Al(OPri)4}−, {Zr2(OPri)9}− and {M(OPri)6}− Units (M = Nb or Ta)

Chloride isopropoxymetallates of cobalt(II), [ClCo{Al(OPrⁱ)₄}], [ClCo{Zr₂(OPrⁱ)₉}] and [ClCo{M(OPrⁱ)₆}] (M = Nb or Ta) have been synthesized by reactions of CoCl₂ with Li{Al(OPrⁱ)₄}, K{Zr₂(OPrⁱ)₉} and K{M(OPrⁱ)₆} in equimolar ratio. The chlorine in these chloride bimetallic isopropoxides has been replaced with alkoxide groups by their reactions with potassium alkoxides yielding products of the types, [(OR)Co{Ta(OPrⁱ)₆}] and [(OR)Co{Zr₂(OPrⁱ)₉}] (R=Me, Prⁿ, Prⁱ, Buⁿ, Bu^s or Bu^t). Alcohol interchange reactions of the above derivatives have been studied. All these new bimetallic alkoxides of cobalt(II) have been characterized by elemental analyses, IR, electronic spectral (visible) and magnetic susceptibility measurements.     

Synthesis,reactions, spectral and magnetic studies of bimetallic alkoxides of cobalt(II) with zirconium(IV)

A bimetallic isopropoxide of cobalt(II) with the formula Co[Zr₂(OPrⁱ)₉]₂, prepared by the reaction of COCl₂ with K[Zr₂(OPrⁱ)₉] in 1:2 molar ratio, has been shown to undergo alcoholysis reactions with graded alcohols (primary, secondary and tertiary) to afford products of the types, Co[Zr₂(OR)₉]₂ (where R = Me, Et, Prⁿ, Buⁿ, Buⁱ and Bu^s) Co[Zr₂(OPrⁱ)₃(OEt)₆]₂, Co[Zr₂(OPrⁱ)₆(OBu^s)₃]₂, CO[Zr₂(OPrⁱ)₃(OBu^s)₆]₂, Co[Zr₂(OPrⁱ)₆(OBu^t)₃]² and Co[Zr₂(OPrⁱ)₃(OR)₆]₂ (where R = Am^t or Bu^t). These derivatives have been characterized by elemental analyses and molecular weight determinations. Infrared, electronic (visible) spectral and magnetic susceptibility measurements suggest a distorted octahedral geometry for these derivatives.     

Carvacrol-induced [Ca2+]i rise and apoptosis in human glioblastoma cells

AimsThis study examined whether the essential oil component carvacrol altered cytosolic free Ca²⁺ level ([Ca²⁺]_i) and viability in human glioblastoma cells.Main methodsThe Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺]_i. Cell viability was measured by detecting reagent WST-1. Apoptosis and reactive oxygen species (ROS) were detected by flow cytometry.Key findingsCarvacrol at concentrations of 400–1000 μM induced a [Ca²⁺]_i rise in a concentration-dependent fashion. The response was decreased partially by removal of extracellular Ca²⁺. Carvacrol-induced Ca²⁺ signal was not altered by nifedipine, econazole, SK&;F96365, and protein kinase C activator phorbol myristate acetate (PMA), but was inhibited by the protein kinase C inhibitor GF109203X. When extracellular Ca²⁺ was removed, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) abolished carvacrol-induced [Ca²⁺]_i rise. Incubation with carvacrol also abolished thapsigargin or BHQ-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 abolished carvacrol-induced [Ca²⁺]_i rise. At concentrations of 200–800 μM, carvacrol killed cells in a concentration-dependent manner. This cytotoxic effect was not changed by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N–-tetraacetic acid/acetoxy methyl (BAPTA/AM). Annexin V/propidium iodide staining data suggest that carvacrol (200, 400 and 600 μM) induced apoptosis in a concentration-dependent manner. At concentrations of 200, 400 and 600 μM, carvacrol induced production of ROS.SignificanceIn human glioblastoma cells, carvacrol induced a [Ca²⁺]_i rise by inducing phospholipase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via protein kinase C-sensitive, non store-operated Ca²⁺ channels. Carvacrol induced cell death that might involve ROS-mediated apoptosis.     

Characterisation of [3H]-Darifenacin as a Novel Radioligand for the Study of Muscarinic M3 Receptors

Abstract

Darifenacin, (S)-2-[1-[2,3-dihydrobenzofuran-5-yl]-3-pyrrolidinyl]-2,2-diphenylacetamide, is a novel muscarinic M₃ antagonist. In this study we have compared the binding of [³H]-darifenacin to the five cloned human muscarinic receptors (m₁ - m₅) expressed in CHO cells. [³H]-darifenacin binds with 6 fold higher affinity to m₃ (K_D = 0.33 nmol/l) over m₁ (K_D = 1.6 nmol/l) receptors. There was no specific binding of [³H]-darifenacin to m₂ receptors and specific binding to m₄ and m₅ receptors was insufficient to determine a K_D. Binding of [³H]-darifenacin to m₁ and m₃ was displaced by atropine (m₁ pK_i = 9.36, m₃ pK_i = 9.4), 4-DAMP (m₁ pK_i = 9.04, m₃ pK_i = 9.19), pirenzepine (m₁ pK_i = 8.63, m₃ pK_i = 6.85), methoctramine (m₁ pK_i = 7.28, m₃ pK_i = 6.63), and darifenacin (m₁ pK_i = 8.36, m₃ pK_i = 9.14), demonstrating that [³H]-darifenacin represents the first selective m₃ radioligand.     

Synthetic,chemical spectroscopic (infrared and electronic) and magnetic studies on dizirconiumenneaisopropoxide complexes of iron(II)

Bimetallic alkoxide derivatives of iron(II) with zirconium of the types [Fe{Zr₂(OPrⁱ)₉}₂], [ClFe- {Zr₂(OPrⁱ)₉}], [(RO)Fe{Zr₂(OPrⁱ)₉}], and [(acac)- Fe{Zr₂(OPrⁱ)₉}] have been synthesized and characterized by elemental analyses, infrared and electronic spectral as well as magnetic susceptibility studies.     

Effect of clotrimazole on cytosolic Ca2+ rise and viability in HA59T human hepatoma cells

Abstract

Clotrimazole is an antimycotic imidazole derivative that interferes with cellular Ca²⁺ homeostasis. This study examined the effect of clotrimazole on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) and viability in HA59T human hepatoma cells. The Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺]_i. Clotrimazole induced [Ca²⁺]_i rises in a concentration-dependent manner. The response was reduced by removing extracellular Ca²⁺. Clotrimazole-evoked Ca²⁺ entry was suppressed by store-operated channel inhibitors (nifedipine, econazole and SK&F96365) and protein kinase C modulators (GF109203X and phorbol, 12-myristate, 13-acetate). In Ca²⁺-free medium, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5-di-tert-butylhydroquinone abolished clotrimazole-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 abolished clotrimazole-induced [Ca²⁺]_i rise. At 10–40?µM, clotrimazole inhibited cell viability, which was not reversed by chelating cytosolic Ca²⁺. Clotrimazole at 10 and 30?µM also induced apoptosis. Collectively, in HA59T cells, clotrimazole-induced [Ca²⁺]_i rises by evoking phospholipase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via store-operated Ca²⁺ channels. Clotrimazole also caused apoptosis.     

Sterically demanding bi- and tridentate alkoxy-N-heterocyclic carbenes

In contrast to the one-pot, two step syntheses we recently reported for a large number of substituted bidentate alkoxy-carbene ligands derived from epoxides, the reaction of imidazole with 2-adamantyl epoxide readily affords the bis(ethoxyadamantyl) substituted imidazolium salt [1-C{(NR)CHCH(NRH)}] (RH = CH₂(2-adamantyl)OH), which has been isolated and structurally characterised as its iodide salt, [HC{(NRH)CH}₂]I. Treatment with group 1 bases results in the loss of one ethoxy arm, to afford the structurally characterised monosubstituted alcohol imidazole, [HC{(NR)CHCHN}], or the lithium carbene complex [LiC{(NR)CHCHN}], a carbene complex containing a singly-N-functionalised alkoxy carbene. Alternatively, the monosubstituted alcohol imidazole may ben requaternised at the nitrogen atom with iso-propyl iodide to form [HC{(NRH)CHCH(NPrⁱ)}]I, from which a more standard lithium alkoxycarbene complex [LiC{(NR)CHCHNPrⁱ}] may be generated.     

Mechanisms of resveratrol-induced changes in [Ca2+]i and cell viability in PC3 human prostate cancer cells

Abstract

Resveratrol is a natural compound that affects cellular Ca²⁺ homeostasis and viability in different cells. This study examined the effect of resveratrol on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and viability in PC3 human prostate cancer cells. The Ca²⁺-sensitive fluorescent dye fura-2 was used to measure [Ca²⁺]_i and WST-1 was used to measure viability. Resveratrol-evoked [Ca²⁺]_i rises concentration-dependently. The response was reduced by removing extracellular Ca²⁺. Resveratrol-evoked Ca²⁺ entry was not inhibited by nifedipine, econazole, SKF96365 and the protein kinase C inhibitor GF109203X, but was nearly abolished by the protein kinase C activator phorbol 12-myristate 13 acetate. In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5-di-tert-butylhydroquinone decreased resveratrol-evoked rise in [Ca²⁺]_i. Conversely, treatment with resveratrol inhibited BHQ-evoked rise in [Ca²⁺]_i. Inhibition of phospholipase C with U73122 did not alter resveratrol-evoked rise in [Ca²⁺]_i. Previous studies showed that resveratrol between 10 and 100?µM induced cell death in various cancer cell types including PC3 cells. However, in this study, resveratrol (1–10?μM) increased cell viability, which was abolished by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid-acetoxymethyl ester (BAPTA/AM). Therefore, it is suggested that in PC3 cells, resveratrol had a dual effect on viability: at low concentrations (1–10?µM) it induced proliferation, whereas at higher concentrations it caused cell death. Collectively, our data suggest that in PC3 cells, resveratrol-induced rise in [Ca²⁺]_i by evoking phospholipase C-independent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry, via protein kinase C-regulated mechanisms. Resveratrol at 1–10?µM also caused Ca²⁺-dependent cell proliferation.     

Synthesis,characterization and inhibitory potency of two oxono and thiono analogues of phosphoramidate compounds on acetylcholinesterase

Two novel structurally related phosphoramidate compounds, 1 and 2, with likely β-diketone system were synthesized and characterized by ¹H, ¹³C, ³¹P NMR, IR spectroscopy and elemental analysis. Compound 2 exhibited a ³¹P NMR signal which was significantly shielded (8 ppm) relative to compound 1. Determination of human erythrocyte acetylcholinesterase (hAChE) inhibitory activity was carried out according to Ellman's modified kinetic method and the IC₅₀ values of compounds 1 and 2 were 1.567 and 2.986 mM, respectively. The k_i values of 1 and 2 were 1.39 to 2.65 min^{? 1} respectively. A comparison of the bimolecular rate constant (k_i) and IC₅₀ values for the irreversible inhibitors 1 and 2 revealed that the oxono analogue has greater affinity for hAChE than the thiono compound. Furthermore effects of two conventional oximes paralidoxime (A) and obidoxime (B) on reactivation of the inhibited hAChE were studied but low reactivity was shown by both the oximes.     

Synthesis and physico-chemical studies of bimetallic alkoxy derivatives of chromium(III) with niobium(V) and tantalum(V)

Reactions of CrCl₃·3thf with KM(OPrⁱ)₆ in 1:3 molar ratios in benzene yield soluble complexes of the type Cr[M(OPrⁱ)₆]₃ (M=Nb or Ta). On heating under vacuum, these complexes tend to disproportionate into Cr(OPrⁱ)₃ and M(OPrⁱ)₅ (M=Nb or Ta). A number of bimetallic alkoxides have also been synthesized by the alcoholysis of Cr[M(OPrⁱ)₆]₃ with alcohols (methanol, ethanol and t-amyl alcohol). The IR, visible, electron spin resonance and magnetic properties of these newly synthesized complexes throw light on the structural features.     

N,N-diisopropylcarboxylic acid amide complexes of thorium(IV) and uranium(IV) N-thiocyanates; the crystal structure of tetraisothiocyanato tetrakis(N,N-diisopropylacetamide-O)uranium(IV)

The complexes M(NCS)₄·xL (x = 2, M = U, L = Me₃CCON(Prⁱ)₂(dippva); x = 3, M = Th, L = Me₂CHCON(Prⁱ)₂(dipiba) and dippva, M = U, L = EtCON(Pr¹)₂(dippa), dipiba and dippva; x = 4, M = Th, L = MeCON(Prⁱ)₂(dipa), dippa and dipiba, M = U, L = dipa, dippa) and the solvates M(NCS)₄·4dipa·CH₂Cl₂ (M = Th, U) have been prepared. Their i.r. and u.v.-visible (M = U only) spectra are reported. The crystal and molecular structure of U(NCS)₄(dipa)₄· CH₂Cl₂ has been determined by the heavy-atom method from X-ray diffractometer data and refined by least squares to R 0.029 for 1135 independent reflections. The crystal is tetragonal, space group P$\text{4}$2₁c, with Z = 2, a = 15.663(4) and c = 10.512(3) Å. The coordination geometry about the 8-coordinate uranium atom is dodecahedral with the N atoms of the NCS groups occupying the dodecahedral A sites and the ‘dipa’ O atoms the B sites. The bonding distances of UO and UN are 2.363(8), and 2.444(11) Å respectively.     

Organic derivatives of aluminium. Part I. Reactions of alkanolamines with aluminium isopropoxide

Reactions of alkanolamines [R₁R₂NXOH; R₁ = H, CH₃, C₂H₅; R₂ = H, CH₃, C₂H₅ and X = -CH₂CH₂-, -CH₂CH₂CH₂-, -CH₂CHCH₃, -C₆H₄CH₂CH₂-] with aluminium isopropoxide in different molar ratios (1 to 3) yield compounds of the type Al(OPrⁱ)_3?n(OXNR₁R₂)_n, where ‘n’ can be 1, 2 and 3. Most of the derivatives are distillable liquids, soluble in common organic solvents and susceptible to hydrolysis even by atmospheric moisture. The new derivatives are characterized by elemental analysis, IR and ¹H NMR spectra. Molecular weight measurements of Al(OPrⁱ)_3?n(OXNR₁R₂)_n reveal them to be tetrameric in nature.     

Characterization and Distribution of a Cloned Rat μ-Opioid Receptor

Abstract: We have cloned and expressed a rat brain cDNA, TS11, that encodes a μ-opioid receptor based on pharmacological, physiological, and anatomical criteria. Membranes were prepared from COS-7 cells transiently expressing TS11 bound [³H]diprenorphine with high affinity (K_D = 0.23 ± 0.04 nM). The rank order potency of drugs competing with [³H]diprenorphine was as follows: levorphanol (K_i = 0.6 ± 0.2 nM) ≈β-endorphin (K_i = 0.7 ± 0.5 nM) ≈ morphine (K_i = 0.8 ± 0.5 nM) ≈ [d -Ala², N-Me-Phe⁴,Gly-ol⁵]-enkephalin (DAMGO; K_i = 1.6 ± 0.5 nM) ? U50,488 (K_i = 910 ± 0.78 nM) > [d -Pen^2,5]-enkephalin (K_i = 3,170 ± 98 nM) > dextrorphan (K_i = 4,100 ± 68 nM). The rank order potencies of these ligands, the stereospecificity of levorphanol, and morphine's subnanomolar K_i are consistent with a μ-opioid binding site. Two additional experiments provided evidence that this opioid-binding site is functionally coupled to G proteins: (a) In COS-7 cells 50 µM 5′-guanylylimidodiphosphate shifted a fraction of receptors with high affinity for DAMGO (IC₅₀ = 3.4 ± 0.5 nM) to a lower-affinity state (IC₅₀ = 89.0 ± 19.0 nM), and (b) exposure of Chinese hamster ovary cells stably expressing the cloned μ-opioid receptor to DAMGO resulted in a dose-dependent, naloxone-sensitive inhibition of forskolin-stimulated cyclic AMP production. The distribution of mRNA corresponding to the μ-opioid receptor encoded by TS11 was determined by in situ hybridization to brain sections prepared from adult female rats. The highest levels of μ-receptor mRNA were detected in the thalamus, medial habenula, and the caudate putamen; however, significant hybridization was also observed in many other brain regions, including the hypothalamus.     

Syntheses and structures of vinyl and aryl derivatives of carbonyl halide iron complexes

cis,trans-Fe(CO)₂(PMe₃)₂(p-Y-C₆H₄)X [X=Br, Y=H (4a), MeO (4b), Cl (4c), F (4d), Me (4e); X=I, Y=H (5); X=Cl, Y=H (6)] and cis,trans-Fe(CO)₂(PMe₃)₂(σ-CHCH₂)X [X=Br (7); X=I (8); X=Cl (9)] are prepared by reacting dihalide complexes cis,trans,cis- Fe(CO)₂(PMe₃)₂X₂ [X=Br (1), X=I (2), X=Cl (3)] with Grignard reagents p-Y-C₆H₄-MgBr (Y=H, OMe, Cl, F, Me) or CH₂CH-MgBr and with lithium reagents PhLi, CH₂CH-Li. With both reagents, the reaction proceeds following two parallel pathways: one is the metallation reaction which yields alkyl derivatives, the other affords 17 electron complexes [Fe(CO)₂(PMe₃)₂X] via monoelectron reductive elimination. The influence of the halides and organometallic reagents on the yield of the metallation reaction is discussed. The solution structure of the complexes is assigned on the basis of IR and ¹H, ¹³C, ¹⁹F, ³¹P NMR spectra. The solid state structure of complexes 4a, 5 and 6 is determined by single crystal X-ray diffractometric methods.     

Structural characterization of zirconium isopropoxide precursors modified by di- and trichloroacetic acids

Reactions of Zr(OⁱPr)₄(PrⁱOH) with di- and trichloroacetic acid in 1:1 molar ratio in toluene gave the products Zr₂(μ-OⁱPr)₂(μ-OOCCHCl₂)(OⁱPr)₄(OOCCHCl₂)(HOⁱPr) (1) and Zr₂(μ-OⁱPr)₂(μOOCCCl₃)(OⁱPr)₄(OOCCCl₃)(HOⁱPr) (2), respectively, in quantitative yields. The molecular geometry of both (1) and (2) is constituted by a slightly distorted edge-shared bioctahedron and both have almost similar bond dimensions. Addition of dichloroacetic acid in 1:2 molar ratio to Zr(OⁱPr)₄(HOⁱPr) in toluene although yielded the bis-substituted crude product Zr(OⁱPr)₂(OOCCHCl₂)₂(HOⁱPr) (3) but its solution in toluene left for crystallization formed a tri-nuclear oxo product Zr₃(μ₃-O)(μ-OⁱPr)₂(μ-OOCCHCl₂)₃(η-OOCCHCl₂)₂(OⁱPr)₃ (3a). The three zirconium atoms in the structure of (3) are forming an isosceles triangle with a triply bridged oxo moiety at its center.     

Monitoring Receptor Mediated Regulation of Cytosolic Calcium in Single Pituitary Cells by Dual Excitation Microfluorimetry

Abstract

Receptor-mediated alterations in the cytosolic free calcium concentration, [Ca²⁺]_i are monitored with the intracellular fluorescent calcium probe fura 2 by dual excitation microfluori-metry. The calcium dependence on the excitation spectrum of fura 2 allows us to choose two wavelengths, λ₁ and λ₂, at which an increase in [Ca²⁺]_i causes either a rise or a fall in fluorescence; the ratio of fluorescence at λ₁ and λ₂ (R =Fλ₁/Fλ₂) is a measure of [Ca²⁺]_i. It appears essential for such measurements that the alteration between the two excitation wavelengths is done rapidly, to allow us to distinguish between effects on [Ca²⁺]_i and other effects on fluorescence. In addition, specific problems relating to the calibration of fura 2 measurements, such as its relative insensitivity at basal [Ca²⁺]_i, the role of intracellular viscosity on fura 2 fluorescence, and the difficulties encountered in establishing calibration constants have to be appreciated. In spite of these potential drawbacks, it appears that monitoring receptor-mediated [Ca²⁺]_i regulation in single cells will prove essential for the further comprehension of stimulus-secretion coupling in pituitary and probably many other cell types.