Showing papers on "Fluorescence spectrometry published in 1992"

Calcium gradients and buffers in bovine chromaffin cells

Abstract: 1. Digital imaging and photometry were used in conjunction with the fluorescent Ca2+ indicator, Fura-2, to examine intracellular Ca2+ signals produced by depolarization of single adrenal chromaffin cells. 2. Depolarization with a patch pipette produced radial gradients of Ca2+ within the cell, with Ca2+ concentration highest in the vicinity of the plasma membrane. These gradients dissipated within a few hundred milliseconds when the voltage-gated Ca2+ channels were closed. 3. Dialysis of Fura-2 into the chromaffin cell caused concentration-dependent changes in the depolarization-induced Ca2+ signal, decreasing its magnitude and slowing its recovery time course. These changes were used to estimate the properties of the endogenous cytoplasmic Ca2+ buffer with which Fura-2 competes for Ca2+. 4. The spatially averaged Fura-2 signal was well described by a model assuming fast competition between Fura-2 and an endogenous buffer on a millisecond time scale. Retrieval of calcium by pumps and slow buffers occurs on a seconds-long time scale. No temporal changes indicative of buffers with intermediate kinetics could be detected. 5. Two independent estimates of the capacity of the fast endogenous Ca2+ buffer suggest that 98-99% of the Ca2+ entering the cell normally is taken up by this buffer. This buffer appears to be immobile, because it does not wash out of the cell during dialysis. It has a low affinity for Ca2+ ions, because it does not saturate with 1 microM-Ca2+ inside the cell. 6. The low capacity, affinity and mobility of the endogenous Ca2+ buffer makes it possible for relatively small amounts of exogenous Ca2+ buffers, such as Fura-2, to exert a significant influence on the characteristics of the Ca2+ concentration signal as measured by fluorescence ratios. On the other hand, even at moderate Fura-2 concentrations (0.4 mM) Fura-2 will dominate over the endogenous buffers. Under these conditions radiometric Ca2+ concentration signals are largely attenuated, but absolute fluorescence changes (at 390 nm) accurately reflect calcium fluxes.

Ultraviolet laser-induced fluorescence of colonic tissue: basic biology and diagnostic potential.

Abstract: Laser-induced fluorescence (LIF) of colonic tissue was examined both in vitro and in vivo to assess the ability of the technique to distinguish neoplastic from hyperplastic and normal tissue and to relate the LIF spectra to specific constituents of the colon. Spectra from 86 normal colonic sites, 35 hyperplastic polyps, 49 adenomatous polyps, and 7 adenocarcinomas were recorded both in vivo and in vitro. With 337-nm excitation, the fluorescence spectra all had peaks at 390 and 460 nm, believed to arise from collagen and NADH, and a minimum at 425 nm, consistent with absorption attributable to hemoglobin. The spectra of colonic tissue recorded both in vivo and in vitro are different, primarily in the NADH fluorescence component, which decays exponentially with time after resection. When normal colonic tissue is compared to hyperplastic or adenomatous polyps, the predominant changes in the fluorescence spectra are a decrease in collagen fluorescence and a slight increase in hemoglobin reabsorption. A multivariate linear regression (MVLR) analysis was used to distinguish neoplastic tissue from non-neoplastic tissue with a sensitivity, specificity, predictive value positive, and predictive value negative toward neoplastic tissue of 80%, 92%, 82%, and 91%, respectively. When the MVLR technique was used to distinguish neoplastic polyps from non-neoplastic polyps, values of 86%, 77%, 86%, and 77% respectively, were obtained. The data suggest that the LIF measurements sense changes in polyp morphology, rather than changes in fluorophores specific to polyps, and it is this change in morphology that leads indirectly to discrimination of polyps.

Calcium transients evoked by climbing fiber and parallel fiber synaptic inputs in guinea pig cerebellar Purkinje neurons.

Abstract: 1. Calcium transients related to climbing fiber (CF) and parallel fiber (PF) synaptic potentials were recorded from Purkinje cells in guinea pig cerebellar slices. Transients were measured using either absorbance changes of arsenazo III or fluorescence changes of fura-2, which were injected into individual cells in the slice. 2. All-or-none somatically recorded CF potentials elicited by white matter stimulation had all-or-none Ca transients. These signals began with a delay of > or = 2 ms from the start of the electrically recorded synaptic potential. The recovery time of CF-induced arsenazo III absorbance transients was < 50 ms in the fine dendrites in conditions that minimized the effects of dye buffering. 3. Ca2+ entry through voltage-gated Ca channels opened by Ca action potentials was the dominant source of the rise in [Ca2+]i after CF activation. There was no significant change in [Ca2+]i corresponding to the plateau potential that followed the large CF response. 4. The appearance and amplitude of distal CF-evoked Ca signals was more variable than proximal signals, suggesting that CF potentials do not reliably spread to the fine distal dendrites. The distal transient could be enhanced by intrasomatic depolarizing pulses, suggesting that it was a property of the postsynaptic membrane and not the presynaptic side of the CF synapse that was responsible for this variability. 5. Parallel fiber responses were evoked by electrical stimulation near the pial surface. Graded synaptic potentials and related Ca transients were reversibly blocked by 2 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Small synaptic potentials induced small, localized Ca transients. With increasing stimulus intensity, the PF electrical response developed a regenerative component. Larger dendritic Ca transients were detected corresponding to this component. Ca transients evoked by the regenerative responses had the same rapid rise times and fall times as those related to somatically stimulated Ca action potentials, suggesting that they also were due to Ca2+ entry through voltage-sensitive channels. 6. During trains of PF responses, we observed an increase in the spatial extent of related Ca transients. This effect could be modulated by changes in the resting potential, suggesting that the same intrinsic mechanism was affecting the spread of both CF and PF signals.

Relative mitochondrial membrane potential and [Ca2+]i in type I cells isolated from the rabbit carotid body.

Abstract: 1. In the accompanying paper (Duchen & Biscoe, 1992) we have described graded changes in autofluorescence derived from mitochondrial NAD(P)H in type I cells of the carotid body in response to changes of PO2 over a physiologically significant range. These observations suggest that mitochondrial function in these cells is unusually sensitive to oxygen and could play a role in oxygen sensing. We have now explored further the relationships between hypoxia, mitochondrial membrane potential (delta psi m) and [Ca2+]i. 2. The fluorescence of Rhodamine 123 (Rh 123) accumulated within mitochondria is quenched by delta psi m. Mitochondrial depolarization thus increases the fluorescence signal. Blockade of electron transport (CN-, anoxia, rotenone) and uncoupling agents (e.g. carbonyl cyanide p-trifluoromethoxy-phenylhydrazone; FCCP) increased fluorescence by up to 80-120%, while fluorescence was reduced by blockade of the F0 proton channel of the mitochondrial ATP synthase complex (oligomycin). 3. delta psi m depolarized rapidly with anoxia, and was usually completely dissipated within 1-2 min. The depolarization of delta psi m with anoxia (or CN-) and repolarization on reoxygenation both followed a time course well characterized as the sum of two exponential processes. Oligomycin (0.2-2 micrograms/ml) hyperpolarized delta psi m and abolished the slower components of both the depolarization with anoxia and of the subsequent repolarization. These data (i) illustrate the role of the F1-F0 ATP synthetase in slowing the rate of dissipation of delta psi m on cessation of electron transport, (ii) confirm blockade of the ATP synthetase by oligomycin at these concentrations, and (iii) indicate significant accumulation of intramitochondrial ADP during 1-2 min of anoxia. 4. Depolarization of delta psi m was graded with graded changes in PO2 below about 60 mmHg. The stimulus-response curves thus constructed strongly resemble those for [Ca2+]i and NAD(P)H with PO2. The change in delta psi m closely followed changes in PO2 with time. 5. The rate of rise of [Ca2+]i in response to anoxia is strongly temperature sensitive. The rate of depolarization of delta psi m with anoxia similarly increased at least two- to fivefold on warming from 22 to 36 degrees C. The change with FCCP was not significantly altered by temperature. 6. These data show that the mitochondrial membrane potential changes over a physiological range of PO2 values in type I cells. This contrasts with the behaviour in dissociated chromaffin cells and sensory neurons, in which no change was measurable until the PO2 fell close to zero.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium influx and calcium current in single synaptic terminals of goldfish retinal bipolar neurons.

Abstract: 1. The calcium influx pathway in large synaptic terminals of acutely isolated bipolar neurons from goldfish retina was characterized using Fura-2 measurements of intracellular calcium and patch-clamp recordings of whole-cell calcium current. 2. Depolarization of bipolar cells with high [K+]o resulted in a sustained, reversible increase in [Ca2+]i in both synaptic terminals and somata. Removal of external calcium abolished the response, as did the addition of 200 microM-cadmium to the bathing solution, indicating that the rise in [Ca2+]i was due to entry of external calcium. Dihydropyridine blockers of voltage-gated Ca2+ channels also blocked the influx, and the Ca2+ channel agonist Bay K 8644 potentiated influx, implicating voltage-activated, dihydropyridine-sensitive channels in the influx pathway. 3. Under voltage clamp, depolarization from a holding potential of -60 mV evoked a slowly inactivating inward current that began to activate at -50 to -40 mV and reached a maximal amplitude between -20 and -15 mV. This current was identified as a calcium current because it decreased when the extracellular calcium concentration was lowered, increased when barium was the charge carrier, and was blocked by 200 microM-external cadmium. The current was substantially blocked by 1 microM-nitrendipine and potentiated by 0.1 microM-Bay K 8644, as expected for L-type Ca2+ channels; it was unaffected by omega-conotoxin. No evidence for transient or rapidly inactivating Ca2+ current was found. 4. At a given level of potassium depolarization, both the amplitude and the speed of increase in [Ca2+]i were greater in synaptic terminals than in somata. For instance, depolarization by 32.6 mM-potassium caused an increase in intracellular calcium of 400 +/- 23 nM in terminals and 180 +/- 20 nM in somata (mean +/- S.E.M., n = 73 terminals, n = 30 somata), with maximal rates of change of 40 +/- 3 and 12 +/- 2 nM/s, respectively. 5. The contribution of terminal and somatic currents to the total whole-cell Ca2+ current was determined under voltage clamp by local application of calcium or of blocking agents. While there was no qualitative difference between currents in terminals and somata, synaptic terminals accounted for 64 +/- 3% (mean +/- S.E.M., n = 12) of the total whole-cell calcium current, and somata accounted for 39 +/- 2%. Thus, the density of Ca2+ current was higher in the terminal, accounting for the greater magnitude and speed of Ca2+ influx observed in terminals in Fura-2 experiments.(ABSTRACT TRUNCATED AT 400 WORDS)

Raman, fluorescence, and time-resolved light scattering as optical diagnostic techniques to separate diseased and normal biomedical media.

Abstract: Studies of Raman scattering, fluorescence and time-resolved light scattering were conducted on cancer and normal biomedical media. Fourier transform Raman spectroscopic measurements were performed on human normal, benign and cancerous tissues from gynecological (GYN) tracts. A comparison of the intensity differences between various Raman modes as well as the number of Raman lines, enables one to distinguish normal GYN tissues from diseased tissues. Fluorescence spectroscopic measurements on human breast tissues show that the ratio of fluorescence intensity at 340 nm to that at 440 nm can be used to distinguish between cancerous and non-cancerous tissues. Separate studies on normal and cancerous breast cell lines show spectral differences. The measurements of back-scattered ultrafast laser pulses from human breast tissues show differences in the scattered pulse profiles for different tissues. These studies show that various optical techniques have the potential to be used in medical diagnostic applications.

Calcium transients in cerebellar Purkinje neurons evoked by intracellular stimulation

Abstract: 1. Purkinje cells in thin slices from the guinea pig cerebellum were injected with fura-2 and high-speed sequences of fluorescence images from the cell body and entire dendritic tree were made while simultaneously recording somatic membrane potential during evoked and spontaneous electrical activity. The changes in fluorescence were interpreted in terms of changes in [Ca2+]i. 2. Individual calcium action potentials were usually accompanied by transient increases in [Ca2+]i all over the dendritic field. During evoked or spontaneous bursts of calcium spikes, [Ca2+]i increased more rapidly and to higher concentrations in fine dendrites than in thicker dendrites. At the end of a burst [Ca2+]i declined faster in thin dendrites than in thicker ones. These variations are most easily understood as deriving from the difference in surface-to-volume ratio of the two kinds of dendrites. 3. During bursts of calcium action potentials [Ca2+]i increases sometimes occurred only in individual dendritic branches, but always including the fine dendrites of that particular branch, showing that calcium action potentials can be regenerative in restrictive parts of the dendritic field without involving the soma or dendritic shaft. 4. Plateau or subthreshold potential changes evoked in the presence of tetrodotoxin (TTX) caused small, widespread increases in [Ca2+]i. The amplitude of these changes was much less than the increase corresponding to spike bursts. The distribution of these plateau Ca signals in thick and thin dendrites was similar to Ca spike-evoked signals, suggesting that the Ca conductances underlying these two potentials are the same or are distributed similarly in the dendrites. 5. Significant increases in [Ca2+]i in the soma were recorded during bursts of sodium-dependent action potentials in normal Ringer. Although much of this increase is due to calcium entry through calcium channels, some of this increase could be due to calcium entry through sodium channels.

Early steps in cytochrome c folding probed by time-resolved circular dichroism and fluorescence spectroscopy

Abstract: The kinetics of protein folding for horse ferricytochrome c was investigated by stopped-flow methods, using far-UV circular dichroism (CD), near-UV CD, and tryptophan fluorescence to probe the formation of secondary structure and tertiary interactions. In the far-UV region of the CD spectrum (222 nm), 44% of the total change associated with refolding occurs within the dead time of the stopped-flow experiment, indicating that a significant amount of helical secondary structure is formed in less than 4 ms. The remaining changes in the ellipticity at 222 nm occur in two kinetic phases with time constants of about 40 ms and 0.7 s, respectively. In contrast, there is no evidence for rapid changes in the ellipticity at 289 nm: an aromatic CD band, which is indicative of the formation of a tightly packed core, only begins to appear in a 400-ms step and is completed in a final 10-s phase. The fluorescence of a single tryptophan at position 59, which becomes quenched upon folding via nonradiative energy transfer to the heme group, provides complementary information on the condensation of the polypeptide chain during refolding. The fluorescence-detected stopped-flow folding kinetics of ferricytochrome c exhibits a 35% decrease in fluorescence during the dead time, suggesting that a substantial decrease in the average tryptophan-heme distance occurs on a submillisecond time scale. The subsequent fluorescence changes exhibit two prominent phases with time constants of about 20 and 300 ms, followed by a minor 5-s phase. Transient peptide CD spectra measured at different folding times (4 ms to 5 s) show no evidence for non-native elements of secondary structure at any stage of folding. Together with previous pulsed amide proton exchange data measured under identical folding conditions (Roder, Elove, & Englander (1988) Nature 335, 700-7041, the results suggest that during the early stages of folding (<4 ms), a partially condensed intermediate with a fluctuating core is formed that contains a significant amount of helical secondary structure but no stable hydrogen bonds. A second intermediate, populated on the 20-1 00-ms time scale, exhibits stable hydrogen-bonded structure in two helical segments near the chain termini and increased compactness, but the aromatic side chains are still in a dynamic or exposed environment. Other helical segments contribute to the helical character of the far-UV CD spectrum but lack sufficiently stable hydrogen bonding to provide protection against NH exchange.

Hematin as a peroxidase substitute in hydrogen peroxide determinations.

Abstract: Hematin can substitute for horseradish peroxidase (HRP) as the catalyst in the determination of hydrogen peroxide using phenolic substrates such as p-hydroxyphenylacetate or p-cresol. Although the peroxidatic activity of hematin from bovine blood is not as great as HRP in terms of unit iron content, the activity per unit weight is substantially greater. Hematin is 500 times less expensive than HRP per unit peroxidatic activity. In hematin-catalyzed systems, reaction development and fluorescence measurement can both be conducted optimally in the same ammoniacal buffer. Hydroxyalkyl hydroperoxides are rapidly hydrolyzed to H2O2 at this pH and are also determined. On the other hand, for methyl hydroperoxide, hematin exhibits only approximately 10% of the sensitivity exhibited by HRP. Hematin is significantly more stable in solution than HRP. The use of hematin as catalyst and p-cresol as the substrate leads to a particularly inexpensive and sensitive system, permitting a limit of detection (LOD) of 7 nM H2O2 in a flow-injection configuration.

Lateral diffusion and percolation in two-phase, two-component lipid bilayers. Topology of the solid-phase domains in-plane and across the lipid bilayer.

Abstract: Fluorescence recovery after photobleaching (FRAP) has recently been used to examine the percolation properties of coexisting phases in two-component, two-phase phosphatidylcholine bilayers [Vaz, W. L. C., Melo, E. C. C., & Thompson, T. E. (1989) Biophys. J. 56, 869-876]. We now report the use of FRAP to study two additional problems in similar systems. The first is the effect of solid-phase obstacles on the lateral diffusion in the fluid phase. The second is the question of whether or not, in a single bilayer, solid-phase domains in one monolayer are exactly superimposed on solid domains in the apposing monolayer. To address the first problem, the lateral diffusion of N-(7-nitrobenzoxa-2,3-diazol-4-yl)-1-palmitoyl-2-oleoylphosp hatidylethanolamine (NBD-POPE), a probe soluble only in the fluid phase when solid and fluid phases coexist, has been studied in the mixture N-lignoceroyldihydrogalactosylceramide (LigGalCer)/dipalmitoylphosphatidylcholine (DPPC). Percolation of the fluid phase occurs at a high mass fraction of solid phase. This indicates that the solid domains have a centrosymmetric shape, a characteristic which makes this a good experimental system to test theoretical simulations of diffusion in an archipelago. It is shown that agreement between theory and experiment is poor, a result that had already been observed when the obstacles were integral membrane proteins. We develop an effective-medium model for diffusion in two-phase systems which explains both our results and those obtained with integral proteins. The distinctive feature of the model is the consideration of an annular region around the obstacles where the lipids are more ordered than in the bulk fluid phase. The diffusion coefficient is then calculated by extending the free area model to two-phase systems, taking these annuli into account. The second question, the organization of the solid-phase domains across the lipid bilayer, is examined in the systems LigGalCer/DPPC and dimyristoylphosphatidylcholine (DMPC)/distearoylphosphatidylcholine (DSPC) by comparing the diffusion of a fluid-phase-soluble, gel-phase-insoluble lipid derivative which spans the two monolayers of a bilayer (NBD-membrane-spanning-phosphatidylethanolamine, NBD-msPE) with that of a probe which is restricted to a single monolayer. In LigGalCer/DPPC, 20:80, the distribution of solid domains in one of the monolayers is independent of the distribution in the apposing monolayer. In contrast, in DMPC/DSPC, 50:50, the solid domains in one monolayer are exactly superimposed upon the solid domains existing in the apposing monolayer.

Solvent effects on the photochromic reactions of diarylethene derivatives

Abstract: This paper investigates the photochromic reactions of diarylethene with heterocyclic rings and electron-with-drawing substituents in various organic solvents. The photoinduced ring-closure process and the fluorescence properties are strongly depend on solvent polarity. This paper attempts to solve the problem of environmental effects on the reaction pathway in connection with the geometries of the excited state molecules.

Synchronous Fluorescence Spectra of Metal-Fulvic Acid Complexes

Abstract: Synchronous quenching spectra, the difference between synchronous scans of ligand fluorescence before and after metal addition, have distinct and readily identifiable patterns for divalent transition metals [Cu(II), Pb(II), Ni(II), Co(II), Mn(II)], Mg(II), and Al(III) binding to fulvic acid (FA). Quenching spectra of Fe(III) resemble those of other transition metals at pH 5.0 but differ at pH 1.5. Principal component analyses of the spectra acquired at varying total metal concentrations demonstrate that multiple fluorescent binding sites for divalent transition metals are present in FA solutions at PH 7.5.

A naphthyl analog of the aminostyryl pyridinium class of potentiometric membrane dyes shows consistent sensitivity in a variety of tissue, cell, and model membrane preparations

Abstract: The fast potentiometric indicator di-4-ANEPPS is examined in four different preparations: lipid vesicles, red blood cells, squid giant axon, and guinea pig heart. The dye gives consistent potentiometric responses in each of these systems, although some of the detailed behavior varies. In lipid vesicles, the dye displays an increase in fluorescence combined with a red shift of the excitation spectrum upon hyperpolarization. Similar behavior is found in red cells where a dual wavelength radiometric measurement is also demonstrated. The signal-to-noise ratio of the potentiometric fluorescence response is among the best ever recorded on the voltage-clamped squid axon. The dye is shown to be a faithful and persistent monitor of cardiac action potentials with no appreciable loss of signal or deterioration of cardiac activity for periods as long as 2 hr with intermittent illumination every 10 min. These results, together with previously published applications of the dye to a spherical lipid bilayer model and to cells in culture, demonstrate the versatility of di-4-ANEPPS as a fast indicator of membrane potential.

Shear stress-induced calcium transients in endothelial cells from human umbilical cord veins.

Abstract: 1. Changes of the free cytosolic Ca2+ concentration induced by shear stress were measured in Fura-2 acetoxymethyl ester-loaded endothelial cells from human umbilical cord veins. 2. We were able to induce Ca2+ transients in almost every cell by blowing a stream of physiological solution onto a single endothelial cell thereby inducing shear stress between 0 and 50 dyn cm-2. The Ca2+ response could be graded by varying the shear stress, and reached a half-maximal value at a shear stress of 30 dyn cm-2. 3. The shear stress responses critically depended on the extracellular Ca2+ concentration and were absent in a Ca(2+)-free solution. Repetitive application of short pulses of shear stress induced cumulative effects because of the slow decay of the shear stress Ca2+ responses (time constants 82.3 +/- 17.8 s from twenty-five cells). Application of a depolarizing high potassium solution to reduce the driving force for Ca2+ entry decreased the Ca2+ transients in some of the cells. 4. Application of shear stress in the presence of other divalent cations, such as nickel, cobalt or barium, always produced substantial changes in the ratio of the 390/360 nm fluorescence signal, indicating influx of these cations and subsequent quenching of the Fura-2 fluorescence. 5. Shear stress responses in the presence of 10 mM Ca2+ were completely blocked by application of 1 mM La3+. 6. Incubation of the cells with the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) did not alter the shear stress response, but completely blocked histamine-induced Ca2+ transients. 7. Small submaximal shear stress potentiated the Ca2+ transients induced by histamine. 8. We conclude that shear stress-dependent Ca2+ signals are induced by an influx of calcium that is not modulated via protein kinase C and not activated by membrane depolarization. The influx pathway is also permeable to divalent cations such as Ni2+, Co2+ and Ba2+, but is blocked by La3+.

Myoplasmic free Mg2+ concentration during repetitive stimulation of single fibres from mouse skeletal muscle.

Abstract: 1. The role of the myoplasmic free Mg2+ concentration ([Mg2+]i) in fatigue was studied in intact single fibres isolated from mouse skeletal muscle. Fatigue was produced by repeated tetanic stimulation. The fluorescent Mg2+ indicator furaptra was pressure injected into fibres. In vivo calibrations were performed to convert fluorescence signals into [Mg2+]i. 2. [Mg2+]i at rest was 0.78 +/- 0.05 mM (mean +/- S.E.M., n = 14). An increase of the extracellular [Mg2+] from 0.5 to 20 mM resulted in a small elevation of [Mg2+]i (86 microM in 5 min). Removal of extracellular Na+ did not affect [Mg2+]i. An intracellular alkanization of about 0.6 pH units gave a [Mg2+]i reduction of 65 microM. 3. During fatiguing stimulation [Mg2+]i initially remained almost constant and it then suddenly started to rise towards the end of the stimulation period. The onset of the [Mg2+]i rise was always followed by a rapid tension decline. In fatigue [Mg2+]i was approximately twice as high as at rest. 4. Fibres were injected with MgCl2 to study if the rise in [Mg2+]i could explain the tension decline in fatigue. An elevation of [Mg2+]i was accompanied by a tension reduction but the [Mg2+]i for a given tension was generally much higher in rested fibres injected with MgCl2 than in fatigued fibres. Thus the rise in [Mg2+]i as such cannot explain the tension reduction in fatigue. 5. Injection of MgCl2 was also used to assess the intracellular Mg2+ buffering. The mean Mg2+ buffer power (i.e. the ratio of the change in [Mg2+]i to the amount of Mg2+ added) was 0.62. 6. ATP is the quantitatively most important binding site for Mg2+ at rest and ATP breakdown is then a likely source of the [Mg2+]i increase in fatigue. The role of ATP breakdown in the increase of [Mg2+]i was studied with metabolic inhibition: fibres were exposed to iodoacetic acid to inhibit glycolysis and cyanide to inhibit oxidative phosphorylation. The pattern during metabolic inhibition was similar to that observed during fatigue. After remaining almost constant during a lengthy period, [Mg2+]i rose rapidly and this rise preceded a period of rapid tension decline. The fibres thereafter went into rigor and [Mg2+]i stabilized at an elevated level; the mean [Mg2+]i increase in rigor was 1.30 mM. 7. We have used modelling to determine the likely change in the intracellular ATP concentration ([ATP]i) for the observed changes in [Mg2+]i.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytosolic calcium and myofilaments in single rat cardiac myocytes achieve a dynamic equilibrium during twitch relaxation.

Abstract: 1. Single isolated rat cardiac myocytes were loaded with either the pentapotassium salt form or the acetoxymethyl ester (AM) form of the calcium-sensitive fluorescent probe, Indo-1. The relationship of the Indo-1 fluorescence transient, an index of the change in cytosolic calcium [Ca2+]i concentration, to the simultaneously measured cell length during the electrically stimulated twitch originating from slack length at 23 degrees C was evaluated. It was demonstrated that even if the Ca2+ dissociation rate from Indo-1 was assumed to be as slow as 10 s-1, the descending limb ('relaxation phase') of the Indo-1 fluorescence transient induced by excitation under these conditions is in equilibrium with the [Ca2+]i transient. Additionally, the extent of Indo-1 loading employed did not substantially alter the twitch characteristics. 2. A unique relationship between the fluorescence transient and cell length was observed during relaxation of contractions that varied in amplitude. This was manifest as a common trajectory in the cell length vs. [Ca2+]i phase-plane diagrams beginning at the time of cell relengthening. The common trajectory could also be demonstrated in Indo-1 AM-loaded cells. The Indo-1 fluorescence-length relation defined by this common trajectory is steeper than that described by the relation of peak contraction amplitude and peak fluorescence during the twitch contractions. 3. The trajectory of the [Ca2+]i-length relation elicited via an abrupt, rapid, brief (200 ms) pulse of caffeine directly onto the cell surface or by 'tetanization' of cells in the presence of ryanodine is identical to the common [Ca2+]i-length trajectory formed by electrically stimulated contractions of different magnitudes. As the [Ca2+]i and length transients induced by caffeine application or during tetanization in the presence of ryanodine develop with a much slower time course than those elicited by electrical stimulation, the common trajectory is not fortuitous, i.e. it cannot be attributed to equivalent rate-limiting steps for the decrease of [Ca2+]i and cell relengthening. 4. The [Ca2+]i-length relation defined by the common trajectory shifts appropriately in response to perturbations that have previously been demonstrated to alter the steady-state myofilament Ca2+ sensitivity in skinned cardiac fibres. Specifically, the trajectory shifts leftward in response to an acute increase in pH or following the addition of novel myofilament calcium-sensitizing thiadiazinone derivatives; a rightward shift occurs in response to an acute reduction in pH or following the addition of butanedione monoxime.(ABSTRACT TRUNCATED AT 400 WORDS)

Fluorescence measurements of cytoplasmic and mitochondrial sodium concentration in rat ventricular myocytes.

Abstract: 1. The fluorescent Na+ indicator SBFI was incorporated into isolated ventricular myocytes using the acetoxymethyl (AM) ester. 2. The excitation spectrum was found to be shifted about 20 nm in the cell compared to in vitro. In the cell, an increase of [Na+] decreased fluorescence at 380 nm (F380) and had no effect at 340 nm (F340). The ratio (R = F340/F380) was used as a measure of [Na+]i. 3. In vivo calibration of SBFI for [Na+]i was obtained by equilibrating [Na+] across the plasma membrane with a divalent-free solution in the presence of gramicidin D. 4. Selective removal of the surface membrane with saponin or digitonin released only about 50% of the indicator. Following saponin treatment, cyanide or carbonylcyanide m-chlorphenylhydrazone (CCCP) increased the apparent [Na+] measured by the remaining (presumably mitochondrial) SBFI. It is suggested that mitochondrial [Na+] is normally less than cytoplasmic. 5. Attempts to examine the effects of metabolic inhibition on [Na+]i were hampered by changes of autofluorescence due to changes of [NADH]. It is shown that this effect can be corrected for using the isosbestic signal (excited at 340 nm). 6. Inhibition of both aerobic metabolism (with CN-) and glycolysis (glucose removal or iodoacetate) produced a gradual increase of [Na+]i. This began before the resting contracture developed and may (via Na(+)-Ca2+ exchange) account for some of the rise of diastolic [Ca2+]i seen in previous work. The rise of [Na+]i began at about the same time as the decrease of systolic contraction and therefore at a time when [ATP]i had begun to fall.

Partitioning of polycyclic aromatic hydrocarbons to marine porewater organic colloids

Abstract: Fluorescence quenching was used to measure the binding of pyrene and phenanthrene to marine interstitial water organic colloids from Boston Harbor, MA. Both pyrene and phenanthrene were sorbed by porewater colloids (K oc colloid ∼10 5.0 and 10 4.3 , respectively) from a heavily contaminated nearshore site. Pyrene had a significantly lower affinity toward colloids from a cleaner location (K oc colloid ∼10 4.5 ). Sediments from the former site were also observed to be especially effective sorbents for these compounds relative to expectations based on the literature.

Carbene formation, hydrogen migration, and fluorescence in the excited states of dialkyldiazirines

Abstract: Photolysis of 3,3-dimethyldiazirine produces an electronically excited singlet state which decays by at least three competitive pathways: (1) fluorescence, (2) nitrogen extrusion and carbene formation, and (3) α-hydrogen migration and propylene formation concurrent with nitrogen extrusion. The temperature dependence of the fluorescence intensity was determined

Contribution of Ca(2+)-induced Ca2+ release to the [Ca2+]i transients in myocytes from guinea-pig urinary bladder.

Abstract: 1. Smooth muscle cells from guinea-pig urinary bladder were studied at an extracellular Ca2+ concentration ([Ca2+]o) of 3.6 mM and 36 degrees C. Fluorescence of Indo-1 was used to monitor the cytosolic calcium concentration ([Ca2+]i) and its changes ([Ca2+]i transients) induced by step membrane depolarizations. 2. During a 6 s depolarization step from -60 to 0 mV [Ca2+]i increased from a resting 118 +/- 22 nM to 1150 +/- 336 nM and decayed to a sustained level of 295 +/- 62 nM. The experiments were designed to evaluate the contribution of the release of intracellularly stored Ca2+ to components of the depolarization-induced [Ca2+]i transient, i.e. 'phasic', which decayed during a maintained depolarization step, and 'tonic' which constituted the sustained elevation of [Ca2+]i above resting level. 3. A short (1 s) application of 10 mM caffeine mimicked the phasic component. After wash-out of caffeine, the subsequent depolarization induced a [Ca2+]i transient with reduced peak, the degree of suppression depending on the interval between wash-out of caffeine and depolarization. The phasic component of the depolarization and the caffeine-induced [Ca2+]i transients were not additive but saturative. 4. The phasic component was largely abolished in the continuous presence of 10 mM caffeine. It was also abolished by a 10 min cell dialysis of 10 microM ryanodine from the pipette solution and was strongly reduced by dialysis of 5 microM thapsigargin. Changes of the tonic component of the depolarization-induced [Ca2+]i transient were much less pronounced with all three interventions. 5. The tonic component of the depolarization-induced [Ca2+]i transient was increased when [Ca2+]o was elevated briefly before a depolarization close to 0 mV, whereas the phasic component was not significantly changed. Similarly, brief application of 1 microM Bay K 8644 increased the tonic component several-fold without modifying significantly the phasic component. 6. It is concluded that depolarization-induced influx of Ca2+ through L-type Ca2+ channels induces the release of Ca2+ from intracellular caffeine-sensitive stores which constitutes the major part of the phasic component. Ca2+ release superimposes on the effects of Ca2+ influx through L-type Ca2+ channels, the non-inactivating part of which constitutes the tonic component of the [Ca2+]i transient. Since the two processes interact, a dissection by simple subtraction is not possible.