Caffeine interaction with fluorescent calcium indicator dyes.

We report that caffeine, in millimolar concentrations, interacts strongly with four common calcium indicator dyes: mag-fura-2, magnesium green, fura-2, and fluo-3. Fluorescence intensities are either noticeably enhanced (mag-fura-2, fura-2) or diminished (magnesium green, fluo-3). The caffeine-induced changes in the fluorescence spectra are clearly distinct from those of metal ion binding at the indicator chelation sites. Binding affinities for calcium of either mag-fura-2 or magnesium green increased only slightly in the presence of caffeine. Caffeine also alters the fluorescence intensities of two other fluorescent dyes lacking a chelation site, fluorescein and sulforhodamine 101, implicating the fluorophore itself as the interaction site for caffeine. In the absence of caffeine, variation of solution hydrophobicity by means of water/dioxane mixtures yielded results similar to those for caffeine. These observations suggest that hydrophobic substances, in general, can alter dye fluorescence in a dye-specific manner. For the particular case of caffeine, and perhaps other commonly used pharmacological agents, the dye interactions can seriously distort fluorescence measurements of intracellular ion concentrations with metal indicator dyes.     

Intracellular calibration of the fluorescent calcium indicator Fura-2

We present the techniques we have used and the problems we have encountered in our laboratories in the in vivo calibration of the fluorescent Ca2(+)-indicator Fura-2. These techniques include the use of potentiometric methods for the precise control and determination of Ca2+ levels in bathing solutions, in association with methods for the equilibration of internal and external solutions with ionophores (Br-A23187, ionomycin, monensin and nigericin). A by-product of these techniques has been the development of a simple procedure that utilizes Fura-2 as a general indicator of ionized Ca2+ concentrations within the physiological range (pCa 7.5 to 5.5), in other experimental solutions. The major advantages of this relatively simple procedure are that it is (i) rapidly performed, (ii) independent of the total EGTA concentration within each experimental solution, (iii) independent of the absolute EGTA purity, and (iv) unaffected by a large number of potentially interfering cations (i.e. Mg2+, H+, K+, Na+) within the test solutions.     

Measurement of intrasynaptosomal free calcium by using the fluorescent indicator quin-2.

The recently synthesized calcium indicator quin -2 was incorporated into synaptosomes from guinea-pig cerebral cortex following uptake and internal hydrolysis of quin -2 tetra-acetoxymethyl ester. Incubation in physiological media containing 1 mM- or 2 mM-CaCl2 led to equilibrium cytosolic ionized calcium concentrations of 85 +/- 10 nM and 205 +/- 5 nM respectively (mean +/- S.E.M. from eight and eighteen preparations respectively). Cytosolic Ca2+ was elevated following increases in external Ca2+ concentration, plasma membrane depolarization, mitochondrial inhibition, calcium ionophore addition or replacement of external sodium by lithium. Preliminary experiments were performed to assess changes in cytosolic Ca2+ accompanying the release of the neurotransmitter acetylcholine.     

Measurement of ionized calcium in blood platelets with the photoprotein aequorin. Comparison with Quin 2

The Ca2+-sensitive photoprotein aequorin (Mr = 20,000) was introduced into human blood platelets by incubation with 10 mM EGTA and 5 mM ATP. Platelet cytoplasmic and granule contents were retained during the loading procedure, and platelet morphology, aggregation, and secretion in response to agonists were normal after aequorin loading. Luminescence indicated an apparent resting cytoplasmic ionized calcium concentration [( Cai2+]) of 2-4 microM in media containing 1 mM Ca2+ and of 0.8-2 microM in 2-4 mM EGTA. The Ca2+ ionophore A23187 and the enzyme thrombin produced dose-related luminescent signals in both Ca2+-containing and EGTA-containing media. Peak [Cai2+] after A23187 or thrombin stimulation of aequorin-loaded platelets was 2-10 microM, while peak [Cai2+] determined using Quin 2 as the [Cai2+] indicator was at least 1 log unit lower. In platelets loaded with both aequorin and Quin 2, the aequorin signal was delayed but not reduced in amplitude. Aequorin loading of Quin 2-loaded cells had no effect on the Quin 2 signal. Ca2+ buffering by Quin 2 (intracellular concentration greater than 1 mM) is also supported by a reciprocal relationship between [Quin 2] and peak [Cai2+] stimulated by A23187 in the presence of EGTA. Parallel experiments with Quin 2 and aequorin may identify inhomogeneous [Cai2+] in platelets and give a more complete picture of platelet Ca2+ homeostasis than either indicator alone.     

Differential mitochondrial calcium responses in different cell types detected with a mitochondrial calcium fluorescent indicator, mito-GCaMP2

Mitochondrial calcium plays a crucial role in mitochondrial metabolism, cell calcium handling, and cell death. However, some mechanisms concerning mitochondrial calcium regulation are still unknown, especially how mitochondrial calcium couples with cytosolic calcium. In this work, we constructed a novel mitochondrial calcium fluorescent indicator (mito-GCaMP2) by genetic manipulation. Mito-GCaMP2 was imported into mitochondria with high efficiency and the fluorescent signals co-localized with that of tetramethyl rhodamine methyl ester, a mitochondrial membrane potential indicator. The mitochondrial inhibitors specifically decreased the signals of mito-GCaMP2. The apparent K(d) of mito-GCaMP2 was 195.0 nmol/L at pH 8.0 in adult rat cardiomyocytes. Furthermore, we observed that mito-GCaMP2 preferred the alkaline pH surrounding of mitochondria. In HeLa cells, we found that mitochondrial calcium ([Ca(2+)](mito)) responded to the changes of cytosolic calcium ([Ca(2+)](cyto)) induced by histamine or thapasigargin. Moreover, external Ca(2+) (100 μmol/L) directly induced an increase of [Ca(2+)](mito) in permeabilized HeLa cells. However, in rat cardiomyocytes [Ca(2+)](mito) did not respond to cytosolic calcium transients stimulated by electric pacing or caffeine. In permeabilized cardiomyocytes, 600 nmol/L free Ca(2+) repeatedly increased the fluorescent signals of mito-GCaMP2, which excluded the possibility that mito-GCaMP2 lost its function in cardiomyocytes mitochondria. These results showed that the response of mitochondrial calcium is diverse in different cell lineages and suggested that mitochondria in cardiomyocytes may have a special defense mechanism to control calcium flux.     

Solubilization and reconstitution of voltage-dependent calcium channel from bovine cardiac muscle. Ca2+ influx assay using the fluorescent dye Quin2

Highly purified sarcolemmal membranes, prepared from fresh bovine heart left ventricle, were solubilized by n-octyl beta-D-glucopyranoside and reconstituted into proteoliposomes with soybean phospholipids by the detergent-dialysis method. Ca2+ flux into the proteoliposomes was determined using the fluorescent probe Quin2. A membrane potential (negative in the proteoliposome interior) that was created by K+ diffusion mediated by valinomycin accelerated the Ca2+ influx. The voltage-dependent Ca2+ influx was dependent on pretreatment of the sarcolemmal membranes with Bay K 8644 and was inhibited by various calcium antagonists including nicardipine (K0.5 = 4.5.10(-7) M), verapamil (K0.5 = 9.2.10(-9) M), diltiazem (K0.5 = 26.10(-8) M) and omega-conotoxin (K0.5 = 9.5.10(-9) M).     

ICPBCZin: a red emitting ratiometric fluorescent indicator with nanomolar affinity for Zn2+ ions

A new fluorescent Zn²⁺ indicator, namely, ICPBCZin was synthesized and the spectral profile of its free and Zn²⁺ bound forms was studied. The newly synthesized zinc indicator incorporates as chromophore the chromeno [3′,2′:3,4]pyrido[1,2a] [1,3]benzimidazole moiety and belongs to the dicarboxylate-type of zinc probes. The compound is excited with visible light, exhibits high selectivity for zinc in the presence of calcium and other common biological ions, and its Zn²⁺ dissociation constant is 4.0 nM. Fluorescence spectra studies of ICPBCZin indicated a clear shift in its emission wavelength maxima upon Zn²⁺ binding, as it belongs to the class of Photoinduced Charge Transfer (PCT) indicators, along with changes in fluorescence intensity that enable the compound to be used as a ratiometric, visible-excitable Zn²⁺ probe.     

Analysis of calcium binding to alpha-lactalbumin using a fluorescent calcium indicator.

A sensitive and rapid assay of Ca2+ binding to proteins was developed, based on the competition of Ca2+ binding to the protein of interest and fluo-3, a fluorescent Ca2+ indicator. Ca2+ binding to fluo-3 and bovine alpha-lactalbumin was analyzed at ten different pH values and a range of Na+ and K+ concentrations. We demonstrate that the binding constants of alpha-lactalbumin, determined by means of the competition assay and using intrinsic protein fluorescence, are the same within experimental error. The dissociation constant of the alpha-lactalbumin--Ca2+ complex in 50 mM Hepes containing 150 mM Na+ at pH 7.4 and 25 degrees C, was found to be 123 +/- 2 nM and 103 +/- 43 nM when determined by the competition assay and intrinsic protein fluorescence, respectively. Binding of Ca2+ to alpha-lactalbumin did not depend on pH in the range 6.6-8.4 and was differently affected by Na+ and K+. EDTA-agarose, a chelating chromatography material, was synthesized and used to remove Ca2+ from buffer and protein solutions. The total concentration of Ca2+ in 50 mM Hepes, containing 150 mM Na+ at pH 7.4, was lowered to 119 +/- 13 nM and the number of Ca2+ bound/molecule alpha-lactalbumin was lowered to 0.069 +/- 0.006. No interaction between fluo-3 and alpha-lactalbumin could be discerned from spectral analysis and fluorescence anisotropy measurements.     

A chlorotetracycline fluorescent probe--an indicator of calcium ion binding with calcium-binding proteins

It has been found in in vitro experiments that fluorescence intensity of deionized solution containing a chlorotetracycline fluorescent probe increases insignificantly at the addition of calmodulin of S-100 proteins. Subsequent introduction of Ca2+ into the medium results in the pronounced fluorescence increase depending on Ca2+ concentration. Addition of specific protein blockers--W7 (calmodulin inhibitor) and antibodies to S-100 brought about a decrease of fluorescence. In in vivo experiments on chlorotetracycline-stained neurons of Helix Pomatia ganglia subesophageal complex it has been shown that bringing of antibodies to S-100 and calmodulin significantly decreases the fluorescence intensity of these cells. These data suggest that the chlorotetracycline probe is an indicator of calcium ions binding with calcium-binding proteins both in in vitro and in vivo systems.     

Myoplasmic calcium transients in intact frog skeletal muscle fibers monitored with the fluorescent indicator furaptra

Furaptra (Raju, B., E. Murphy, L. A. Levy, R. D. Hall, and R. E. London. 1989. Am. J. Physiol. 256:C540-C548) is a "tri-carboxylate" fluorescent indicator with a chromophore group similar to that of fura-2 (Grynkiewicz, G., M. Poenie, and R. Y. Tsien. 1985. J. Biol. Chem. 260:3440-3450). In vitro calibrations indicate that furaptra reacts with Ca2+ and Mg2+ with 1:1 stoichiometry, with dissociation constants of 44 microM and 5.3 mM, respectively (16-17 degrees C; ionic strength, 0.15 M; pH, 7.0). Thus, in a frog skeletal muscle fiber stimulated electrically, the indicator is expected to respond to the change in myoplasmic free [Ca2+] (delta[Ca2+]) with little interference from changes in myoplasmic free [Mg2+]. The apparent longitudinal diffusion constant of furaptra in myoplasm was found to be 0.68 (+/- 0.02, SEM) x 10(-6) cm2 s-1 (16-16.5 degrees C), a value which suggests that about half of the indicator was bound to myoplasmic constituents of large molecular weight. Muscle membranes (surface and/or transverse-tubular) appear to have some permeability to furaptra, as the total quantity of indicator contained within a fiber decreased after injection; the average time constant of the loss was 302 (+/- 145, SEM) min. In fibers containing less than 0.5 mM furaptra and stimulated by a single action potential, the calibrated peak value of delta[Ca2+] averaged 5.1 (+/- 0.3, SEM) microM. This value is about half that reported in the preceding paper (9.4 microM; Konishi, M., and S. M. Baylor. 1991. J. Gen. Physiol. 97:245-270) for fibers injected with purpurate-diacetic acid (PDAA). The latter difference may be explained, at least in part, by the likelihood that the effective dissociation constant of furaptra for Ca2+ is larger in vivo than in vitro, owing to the binding of the indicator to myoplasmic constituents. The time course of furaptra's delta[Ca2+], with average values (+/- SEM) for time to peak and half-width of 6.3 (+/- 0.1) and 9.5 (+/- 0.4) ms, respectively, is very similar to that of delta[Ca2+] recorded with PDAA. Since furaptra's delta[Ca2+] can be recorded at a single excitation wavelength (e.g., 420 nm) with little interference from fiber intrinsic changes, movement artifacts, or delta[Mg2+], furaptra represents a useful myoplasmic Ca2+ indicator, with properties complementary to those of other available indicators.     

The interaction of calcium and magnesium ions with deoxyribonucleic acid.

Conductance and equilibrium dialysis studies are reported for the aqueous systems (native calf thymus) DNA-CaCl₂ and DNA-MgCl₂ at various pH values and ionic strengths at 25 °C. Discontinuities occur in the conductance curves at mole ratios of Ca²⁺ and Mg²⁺ to nucleate phosphorus of 0.125, 0.30, and 0.50. The dialysis results show the formation of complexes of stoichiometry 0.50 and 1.00 mol Ca²⁺ or Mg²⁺/mol nucleate phosphorus (2:1 and 1:1 complexes), the latter only in neutral or alkaline solutions, in agreement with the conductance discontinuity at 0.50. The other discontinuities may be due to preferential binding in the formation of the 2:1 complex. Binding constants for the 2:1 complexes are evaluated. Absorption-temperature profiles have been determined for “native” and dialysed DNA in the presence of NaCl, CaCl₂, and MgCl₂. For dialysed DNA at 26 ° C and 260 nm the decrease of absorbance with increased salt concentration was halted for MgCl₂ and CaCl₂ at a concentration corresponding to the formation of the 2:1 complex. The absorbance of “native” DNA did not decrease. T_m and the reciprocal of the hypochromic rise ($\text{1}\text{h}$) increased linearly with log (salt concn). Values of T_m were the same at 230, 260, and 280 nm, but h was greater at 230 and 280 than at 260 nm, which may be due to the existence of alternating blocks of (A + T) and (G ? C) pairs. The entropy of transition was in the order Ca > Mg ? Na.     

Formation constants for interaction of citrate with calcium and magnesium ions

Formation constants for the interaction of citrate ion with calcium and magnesium ions in solution at 37°C and a constant ionic strength of 0.15 were determined by potentiometric titration. Values for the formation of CaL^? and CaHL⁰ complexes were 1.88 × 10³ and 67, respectively. Corresponding constants for MgL^? and MgHL⁰ were 2.19 × 10³ and 42, respectively. The existence of other complexes was not confirmed. Protonation constants were also determined under the same conditions.     

Stimulus-response coupling in human platelets. Changes evoked by platelet-activating factor in cytoplasmic free calcium monitored with the fluorescent calcium indicator quin2.

Rat pancreatic islets contain a Ca2+-activated and thiol-dependent transglutaminase (EC 2.3.2.13) comparable in activity with that found in rat liver, lung and spleen. The Ca2+-dependence of this enzyme is such that half-maximal velocity was obtained in the region of 40 microM. Preincubation of rat islets with primary-amine substrates of transglutaminase (monodansylcadaverine, methylamine, ethylamine, propylamine and cystamine) led to an inhibition of glucose-stimulated insulin release by these amines. Kinetic analysis of the competitive substrates methylamine, monodansylcadaverine, propylamine and ethylamine for their ability to inhibit islet transglutaminase activity indicated a potency that matched their ability to inhibit glucose-stimulated insulin release. When these amines were tested for their effects on glucose-stimulated protein synthesis and glucose utilization, the most potent inhibitor of insulin release, monodansylcadaverine, had no effect on either process at 100 microM. The amines cystamine, ethylamine, methylamine and propylamine had variable effects on these metabolic processes. For ethylamine, methylamine and propylamine, concentrations were found which inhibited glucose-stimulated insulin release in a manner which was found to be independent of their effects on either glucose oxidation or protein synthesis. Primary amines may therefore inhibit insulin release through their incorporation by islet transglutaminase into normal cross-linking sites. A role for protein cross-linking in the secretory mechanism is suggested.     

The interaction of magnesium ions with the calcium pump of sarcoplasmic reticulum.

1. In the presence of Ca2+, ATP phosphorylates the Ca2+ pump of sarcoplasmic reticulum at the same site and to the same extent regardless of whether Mg2+ is added or not to the incubation media, the main effect of added Mg2+ being to increase the rate of phosphorylation. 2. When phosphoenzyme is made in Mg2+-containing media it dephosphorylates about 30-times faster than when it is made in the absence of added Mg2+. Addition of Mg2+ after phosphorylation is uneffective in accelerating the hydrolysis of phosphoenzyme even in solubilized enzyme, suggesting that phosphorylation of the Ca2+ pump results in occlusion of the site at which Mg2+ combines to accelerate the release of phosphate. 3. Occlusion of the site for Mg2+ can be partially reversed by trans-1,2-diaminocyclohexonetetraacetic acid (CDTA). Use was made of this property to demonstrate that for the rapid release of phosphate to occur Mg2+ has to be bound to the enzyme. 4. Results seem to indicate that Mg2+ combines with the Ca2+ pump prior to phosphorylation.     

Na+/Ca2+ exchange in isolated smooth muscle cells demonstrated by the fluorescent calcium indicator fura-2

Fura-2, a novel fluorescent indicator of cytoplasmic calcium concentrations ([Ca2+i]), was 'loaded' into smooth muscle cells isolated from guinea pig taenia coli. Resting cells maintained a stable [Ca2+i] of 107 +/- 26 nM (n = 13), which could be perturbed with ionomycin. [Ca2+i] was elevated by stimulation of the cells with carbachol or 50 mM KCl. Reduction of the plasmalemmal Na+ concentration gradient by inhibition of the Na+/K+-ATPase with ouabain markedly elevated [Ca2+i]; this elevation was dependent on extracellular Ca2+. [Ca2+i] was also increased by replacement of the extracellular Na+ with an organic cation.     

Studies on interaction of oligopeptides with sodium dodecyl sulfate: Stopped-flow kinetics of chemical modification of tryptophan residues withN-bromosuccinimide

The stopped-flow kinetics of the reaction between oligopeptides containing tryptophan residues andN-bromosuccinimide (NBS) were studied in 50 mM sodium phosphate buffer (pH 7.0) containing sodium dodecyl sulfate (SDS). Decreases in the reaction rates attributable to the interaction between oligopeptides and SDS were observed, and oligopeptides studied were classified into types I and II on the basis of the interaction modes. Type I oligopeptides were dissolved in SDS micelles; type II oligopeptides interacted cooperatively with SDS monomers. The manner of interaction between SDS and oligopeptides of type II could be interpreted by a simple equilibrium relation: oligopeptide+n·(SDS)=oligopeptide·(SDS) _n .     

Zinc ions cause the thimerosal-induced signal of fluorescent calcium probes in lymphocytes

Most fluorescent probes for the investigation of calcium signaling also detect zinc ions. Consequently, changes in the intracellular zinc concentration could be mistaken for calcium signals. Thimerosal (TMS) is used as a calcium-mobilizing agent and we analyzed the contribution of zinc ions to the signal observed with fluorescent calcium probes after TMS stimulation. Our findings show that the fluorescent signal in lymphocytes is entirely due to zinc release. Experiments in the T lymphocyte cell line Jurkat and primary human lymphocytes show that TMS and its active metabolite, ethyl mercury, cause an increase in signal intensity with probes designed for the detection of either calcium or zinc ions. The TMS/ethyl mercury-induced signal of the calcium probes Fluo-4 and FURA-2 was completely absent when the zinc chelator TPEN [N,N,N',N'-tetrakis-(2-pyridyl-methyl)ethylenediamine] was added. In contrast, the signal caused by thapsigargin-induced release of calcium from the endoplasmic reticulum was unaffected by TPEN. In light of these observations, zinc may also contribute to calcium signals caused by mercury-containing compounds other than TMS, and a potential involvement of zinc release in the immunomodulatory effects of these substances should be considered.     

Photophysics of the fluorescent Ca2+ indicator Fura-2.

The photophysics of the complex forming reaction of Ca2+ and Fura-2 are investigated using steady-state and time-resolved fluorescence measurements. The fluorescence decay traces were analyzed with global compartmental analysis yielding the following values for the rate constants at room temperature in aqueous solution with BAPTA as Ca2+ buffer: k01 = 1.2 x 10(9)s-1, k21 = 1.0 x 10(11) M-1 s-1, k02 = 5.5 x 10(8) s-1, k12 = 2.2 x 10(7) s-1, and with EGTA as Ca2+ buffer: k01 = 1.4 x 10(9) s-1, k21 = 5.0 x 10(10) M-1 s-1, k02 = 5.5 x 10(8) s-1, k12 = 3.2 x 10(7) s-1. k01 and k02 denote the respective deactivation rate constants of the Ca2+ free and bound forms of Fura-2 in the excited state. k21 represents the second-order rate constant of binding of Ca2+ and Fura-2 in the excited state, whereas k12 is the first-order rate constant of dissociation of the excited Ca2+:Fura-2 complex. The ionic strength of the solution was shown not to influence the recovered values of the rate constants. From the estimated values of k12 and k21, the dissociation constant K*d in the excited state was calculated. It was found that in EGTA Ca2+ buffer pK*d (3.2) is smaller than pKd (6.9) and that there is negligible interference of the excited-state reaction with the determination of Kd and [Ca2+] from fluorimetric titration curves. Hence, Fura-2 can be safely used as an Ca2+ indicator. From the obtained fluorescence decay parameters and the steady-state excitation spectra, the species-associated excitation spectra of the Ca2+ free and bound forms of Fura-2 were calculated at intermediate Ca2+ concentrations.     

Stopped-flow fluorescence studies on binding kinetics of neurotoxins with acetylcholine receptor

Acetylcholine receptor from Narke japonica electroplax exhibits a fluorescence change upon binding of snake neurotoxins. This fluorescence change primarily arises from the conformational change of the acetylcholine receptor and reflects the binding process of the toxin with the receptor. The time dependence of the fluorescence change has been monitored for 28 short neurotoxins and 8 long neurotoxins by using a stopped-flow technique. The steady-state fluorescence change is of the same order of magnitude for the short neurotoxins but varies among the long neurotoxins. Nha 10, a short neurotoxin with weak neurotoxicity, causes no fluorescence change in the receptor but can still bind to the receptor with sufficiently high affinity. The substitution of the conserved residue Asp-31 to Gly-31 in Nha is probably responsible for the reduced neurotoxicity. The rate constants for the binding of the neurotoxins to the receptor have been obtained by analyzing the transient fluorescence change. The rate constants show surprisingly a wide range of distribution: (1.0-20.5) X 10(6) M-1 s-1 for short neurotoxins and (0.26-1.9) X 10(6) M-1 s-1 for long neurotoxins. Examination of the relationship between the rate constants of fluorescence change of the short neurotoxins and their amino acid sequences, thermal stability, hydrogen-deuterium exchange behavior, overall net charge, etc. reveals the following. Positive charges on the side chains of residues 27 and 30 and overall net charge of the neurotoxin govern the magnitude of the binding rate of the neurotoxin with the receptor.