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PubMed1995

Contractile and intracellular Ca2+ decay in potentiated contractions following multiple extrasystolic beats.

Developed pressure and intracellular Ca2+ ([Ca2+i) decay in postextrasystolic beats following multiple extrasystolic contractions (ESCs) was evaluated with surface fluorometry in atrioventricular-blocked perfused rat hearts loaded with Indo-1. After priming pacing at 400 ms intervals, 1-25 ESCs were interposed with a 160 ms interval, followed by 30 postextrasystolic beats with a 400 ms interval. Both left ventricular developed pressure and the amplitude of the Indo-1 fluorescence ratio (F400/F510: an index of [Ca2+]i) increased in a monoexponential manner with an increase in the number of ESCs. Both potentiated left ventricular developed pressure and the amplitude of F400/F510 transients returned to control in a monoexponential fashion. Consistent with this exponential decay, the relationship between developed pressure or the amplitude of F400/F510 transients in a postextrasystolic beat and that in the preceding beat was linear and the slope of a fitted line (recirculation fraction; RF) was evaluated as an index of rapidity of decay. The number of ESCs did not affect RF of developed pressure and the amplitude of F400/F510 transients. Reducing extracellular Ca2+ concentration (1.25 --> 0.55 mM), and perfusion with an acidic solution (pH = 6.8) significantly decreased RF of both developed pressure (0.85 +/- 0.06 --> 0.78 +/- P < 0.05 and 0.85 +/- 0.07 --> 0.78 +/- 0.06, n=8, P < 0.05, respectively) and the amplitude of F400/F510 (0.87 +/- 0.06 --> 0.78 +/- 0.05, P < 0.05, and 0.89 +/- 0.08 --> 0.78 +/- 0.07, P < 0.05, respectively). This study confirmed that, in all conditions evaluated, contractile decay was determined by [Ca2+]i decay and RF of contractile decay was an accurate estimate of [Ca2+]i decay in physiologically paced isolated perfused rat hearts.

Acidosis↗
PubMed1995

Complement component C9-dependent cytosolic free Ca2+ rise and recovery in neutrophils.

The cytosolic free Ca2+ concentration in rat neutrophils was determined by ratiometric fluorometry and imaging of Fura-2. Transient elevations of cytosolic free Ca2+ concentration were provoked by addition of C9 to neutrophils pre-coated with C5b-8. The rate of rise of the cytosolic free Ca2+ concentration was dependent upon the concentration of C9. These changes in cytosolic free Ca2+ concentration occurred in the absence of cell lysis, since there was no release of Fura-2, and were the result of increased permeability to extracellular Ca2+. More than 96% of the rise in cytosolic free Ca2+ generation by C9 was dependent upon the presence of extracellular Ca2+, but did not occur via channels which were inhibited by the Ca2+ channel blocker SKF96365. The decrease in the permeability of the membrane to Ca2+ after C9 was not triggered by the rise in cytosolic free Ca2+. After attack by C9, individual neutrophils remained responsive to f-met-leu-phe, or further attack by C9, both producing Ca2+ transients. The recovery of the Ca2+ signal was consistent with the complement membrane attack complex generating a series of permeability thresholds in the plasma membrane. These data have implications for the understanding of the mechanisms underlying the inappropriate responsiveness of neutrophils at inflammatory sites.

Amino Acids, Diamino↗
PubMed1991

Validation of a toxicity testing model by evaluating oxygen supply and energy state in the isolated perfused rat kidney. Single-pass preparation without albumin.

Validation of a toxicity testing model concerning energy metabolism was attempted by evaluating the oxygen supply and energy state in an isolated perfused rat kidney of single-pass preparation without albumin. Perfusion was performed at a temperature of 31 degrees C, flow rate of 11.0 mL/g/min, and pressure of 81-104 mmHg. The perfusate was saturated with 95% O2/5% CO2. After preperfusion for 30 min, the redox states of cytochrome aa3 and c and pyridine nucleotides (PN) in the perfused kidney were measured to be stable for 90 min by a scanning reflectance spectrophotometry and surface fluorometry, respectively. During the same period, the contents of ATP and inorganic phosphate (Pi) in the perfused kidney were also measured to be stable by 31P-NMR spectroscopy. The oxygen supply to the cell was more than the amount required for the basal metabolism of the cell. For assessment of the effects of chemical agents on the renal cell metabolism, this preparation of the perfused rat kidney was considered to have several advantages, despite some of its inherent limitations in the function of the kidney.

Adenosine Triphosphate↗
PubMed1980

Monoamine metabolites in cerebrospinal fluid of depressive subgroups.

Lumbar punctures were performed on 69 patients who met Research Diagnostic Criteria (RDC) for major affective disorder, while they were drug-free and depressed. None of the patients met RDC for alcoholism. Cerebrospinal fluid 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA) were measured by fluorometry and 3-methoxy-4-hydroxyphenylglycol (MHPG) by gas chromatography. Family histories were ascertained by systematic interviews of patients and their relatives, and diagnoses were made by family history diagnostic criteria (Andreasen et al., 1977). Depressed patients with alcoholism in a first degree relative had significantly lower levels of 5-HIAA and MHPG than patients without a family history of alcoholism (p < 0.05). No difference in HVA levels was found. The metabolite differences remained significant when the influence of sex ratio was considered. These results are in agreement with previous work linking alcoholism to abnormal serotonin metabolism. They provide further biochemical evidence of distinct genetic subtypes of affective disorder along lines suggested by Winokur (1979a, 1979b), and illustrate the usefulness of the family history method in defining patient subgroups.

Adult↗
PubMed1987

Early transmembrane events in tumor necrosis factor and lymphotoxin-induced cytotoxicity.

We have studied early transmembrane events in tumor necrosis factor (TNF) and lymphotoxin (LT)-induced cytotoxicity by means of stopped-flow fluorometry with 3 different fluorescent probes. Both TNF and LT caused an increase in the membrane fluidity of the target cells (L X P3 cells). After this event, there was a low level of calcium influx from the external medium into the target cells. On the other hand, the release of calcium from intracellular stores in the target cells was negligible. These sequential events, however, were not observed when mutant LT molecules which lacked the lytic activity were used.

Aminoquinolines↗
PubMed1994

Developmental alterations in NMDA receptor-mediated [Ca2+]i elevation in substantia gelatinosa neurons of neonatal rat spinal cord.

Using spinal cord slices prepared from neonatal rats, the intracellular free Ca2+ concentration ([Ca2+]i) in neurons located in the dorsal horn substantia gelatinosa (SG) was measured with microscopic fluorometry by loading fura 2-AM into neurons. Developmental alterations in the elevation of [Ca2+]i elicited by the glutamate analogs, NMDA and AMPA, were investigated from postnatal day (PNDs) 1 to 17. During the 1st week of postnatal life, when neuronal maturation of the SG is known to take place, the NMDA response remained large or even slightly increased. It subsequently showed a gradual decline. This pattern of postnatal changes is consistent with previously reported autoradiographic studies on NMDA-binding sites. The affinity of receptors for NMDA was found to decrease constantly during the period examined. The AMPA response and resting [Ca2+]i showed no significant developmental changes. Neonatal treatment with capsaicin, which has been shown to degenerate fine primary afferent fibers terminating in the SG, delayed the developmental decline in the NMDA-induced [Ca2+]i response. It is suggested that the number and the molecular properties of NMDA receptors expressed in the SG change during early postnatal neuronal maturation. The temporal coincidence between postnatal alteration in NMDA-induced [Ca2+]i elevation and neuronal maturation of the SG may indicate that intracellular Ca2+ regulated by NMDA receptor activation is related to postnatal neuronal maturation. Activation of fine primary afferent fibers may contribute to the observed developmental alterations in the NMDA response of SG neurons.

Animals↗
PubMed1986

A comparison of the intrinsic fluorescence of red kangaroo, horse and sperm whale metmyoglobins.

Several metmyoglobins (red kangaroo, horse and sperm whale), containing different numbers of tyrosines, but with invariant tryptophan residues (Trp-7, Trp-14), exhibit intrinsic fluorescence when studied by steady-state front-face fluorometry. The increasing tyrosine content of these myoglobins correlates with a shift in emission maximum to shorter wavelengths with excitation at 280 nm: red kangaroo (Tyr-146) emission maximum 335 nm; horse (Tyr-103, -146) emission maximum 333 nm; sperm whale (Tyr-103, -146, -151) emission maximum 331 nm. Since 280 nm excites both tyrosine and tryptophan, this strongly suggests that tyrosine emission is not completely quenched but also contributes to this fluorescence emission. Upon titration to pH 12.5, there is a reversible shift of the emission maximum to longer wavelengths with an increase greater than 2-fold in fluorescence intensity. With excitation at 305 nm, a tyrosinate-like emission is detected at a pH greater than 12. These studies show that: (1) metmyoglobins, Class B proteins containing both tyrosine and tryptophan residues, exhibit intrinsic fluorescence; (2) tyrosine residues also contribute to the observed steady-state fluorescence emission when excited by light at 280 nm; (3) the ionization of Tyr-146 is likely coupled to protein unfolding.

Animals↗
PubMed1987

Intrinsic fluorescence of carp hemoglobin: a study of the R----T transition.

The intrinsic fluorescence of hemoglobins is known to respond to ligand-induced changes in the quaternary structure of the protein. Carp hemoglobin is an interesting model to study the quaternary transition since its R----T equilibrium is pH-dependent and at low pH, in the presence of organic phosphate, it remains in the T or 'deoxy' quaternary structure, even when saturated with ligand. In this study, using front-face fluorometry, we show that the intrinsic fluorescence intensity exhibited by carp carboxyhemoglobin increases as the pH is lowered below 6.5 in the presence of inositol hexaphosphate. At low pH, carp methemoglobin is less affected by the addition of inositol hexaphosphate than is the CO derivative, while little or no change is observed in the met-azide derivative. We conclude: (1) the exact nature of the R to T state transition induced by inositol hexaphosphate differs for carp carboxy-, met- and met-azide hemoglobin derivatives; (2) the chromophores responsible for the changes observed with absorption spectroscopy may not be the same as those chromophores responsible for the fluorescence differences; and (3) alpha 46-Trp is tentatively assigned as one source of fluorescence emission. Furthermore, fluorescence properties of carp hemoglobin are compared to those of human hemoglobin.

Animals↗
PubMed1987

Dynamic aspects of the heme-binding site in phylogenetically distant myoglobins.

Dynamic aspects of the heme-binding site of myoglobins derived from two phylogenetically distant species, namely sperm whale and bluefin tuna, have been investigated by studying steady-state and time-resolved emission properties of 2-p-toluidinyl-6-naphthalene sulfonic acid (TNS) apomyoglobin conjugates. Multi-frequency phase and modulation fluorometry data indicate that charge movements occur in the fluorophore environment during the excited state lifetime in the sperm whale myoglobin system. In the case of the bluefin tuna myoglobin TNS adduct these movements were not detected, indicating that the relaxation processes differ in the two types of myoglobins.

Animals↗
PubMed1989

Time-resolved fluorescence studies on protoporphyrin IX-apohorseradish peroxidase.

The hemin moiety of horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) was removed and the apoprotein reconstituted with the fluorescent protoporphyrin IX. Steady-state and time-resolved fluorescence properties of the HRP(desFe) adduct were examined; the multifrequency phase and modulation method was utilized for lifetime and dynamic polarization studies. The emission spectrum of HRP(desFe) had maxima at 633 and 696 nm. The lifetime of this emission was characterized by a single exponential decay of 16.87 ns at 22 degrees C. Debye rotational relaxation times for HRP(desFe) were determined using both static (Perrin plot) and dynamic (differential phase and modulation fluorometry) methods; these two approaches gave values of 96 and 86 ns, respectively. A spherical protein of HRP's molecular weight and partial specific volume would be expected to have a Debye rotational relaxation time, at 22 degrees C, in the range of 50 to 60 ns, depending upon the extent of hydration. Hence our results indicate that HRP(desFe) is asymmetric; the global rotational relaxation times observed are consistent with those of a prolate ellipsoid with an axial ratio of 3:1.

Apoenzymes↗
PubMed1991

Analysis of the acid and alkaline dissociation of earthworm hemoglobin, Lumbricus terrestris, by front-face fluorescence spectroscopy.

The steady-state fluorescence properties of the multisubunit hemoglobin isolated from the earthworm, Lumbricus terrestris, were studied by front-face fluorometry. Acid and alkaline dissociation of this high-molecular-weight hemoglobin were examined over the pH range 3.7-12.5 using different liganded states (oxy, CO, met). The relative intensity of the emission maximum at 320 nm (exc. 280 nm) is ligand-dependent increasing as follows: oxy less than deoxy less than CO less than met at pH 7.0. The intensity of the emission maximum of oxyhemoglobin at the alkaline acid end point, pH 10.5 (333 nm), is significantly greater than that observed at the acid end point, pH 4.18 (320 nm), suggesting different subunit dissociation. The spectra of oxyhemoglobin at pH 4.18 and the spectrum of carbonmonoxy hemoglobin at pH 7.0 in the presence of 1 M magnesium chloride were almost identical, indicating similar subunit dissociation. Difference spectrum (pH 9.0-7.2) of fluorescence emission (exc. 305) resulted in a maximum at 341 nm, indicative of tyrosinate formation. This suggests that tyrosine(s) may also be located at the subunit interface(s) of this hemoglobin. These studies indicate that several aromatic amino acid residues are associated with the critical sites of subunit interactions within this molecule. Analysis of the fluorescence spectra also suggests that the formation of different subunit species resulting from acid and alkaline dissociation cannot be ruled out.

Animals↗
PubMed1993

Fluorometric analysis of native, urea-denatured and refolded human prostatic acid phosphatase.

Human prostatic acid phosphatase (EC 3.1.3.2) was denatured in 6 M urea at pH 2.5, but was refolded by dilution at pH 7.0, as demonstrated by the recovery of nearly complete enzyme activity and dimeric structure. The conformational changes among the native, denatured and refolded states were monitored by means of steady-state and nanosecond pulse fluorometry of tryptophan residues of the enzyme. The relative quantum yield of the fluorescence was highest in the native enzyme and lowest in the denatured one, and was intermediate in the refolded enzyme, although the emission peak was reproducible after refolding. The observed decay curves of tryptophan fluorescence of the native, denatured and refolded states were analyzed by decay functions of three lifetimes. The fluorescence lifetimes of the refolded enzyme were shorter than those of the native one. The fluorescence of the denatured enzyme decayed much faster than that of the other forms. The fluorescence excitation spectra revealed that the excitation energy of phenylalanine was transferred to tryptophan(s) in the native and refolded forms, but not in the denatured form. The efficiency of the energy transfer was higher in the native enzyme than in the refolded one. It was found by excitation polarization spectra that the freedom of internal motion of tryptophans was greater in the refolded enzyme than in the native enzyme. In the denatured enzyme the polarization anisotropies were very low. These results indicate that the higher structure with respect to tryptophans of the refolded enzyme is delicately but definitely different from that of the native enzyme and that local conformation of the active center is recovered upon refolding.

Acid Phosphatase↗
PubMed1986

The status of zinc in malaria (Plasmodium falciparum) infected human red blood cells: stage dependent accumulation, compartmentation and effect of dipicolinate.

The inhibitory effect of metal chelators on intraerythrocytic malarial parasites imply that trace metal have a vital role in the biology of these organisms. In the present work X-ray fluorometry was used to study the status of zinc and iron in human red blood cells infected with Plasmodium falciparum in culture conditions. It was found that while the iron level remains constant throughout the parasite cell cycle, that of zinc increases in parallel with parasite maturation to reach a 2.3-fold higher level than that of uninfected red blood cells. Compartment analysis of infected red blood cells indicated that most of this gain was associated with the parasite and some with the host-cell membrane. Analysis of the malarial pigment showed that the zinc/iron ratio was similar to that of red blood cells, implying the this compound, which results from the digestion of host-cell cytosol, sequesters the zinc of host metalloenzymes. Dipicolinic acid (DPA), like other chelators, was found to inhibit the intracellular development of the parasite with an ED50 of 1 mM. DPA does not penetrate into normal red blood cells but readily permeates into infected cells, although it does not leach out their zinc. It is uncertain whether the inhibitory effect of DPA is exerted through alterations of host cell metabolism or by directly affecting that of the parasite. The putative receptors of zinc in the infected red blood cell are discussed.

Animals↗
PubMed1990

Dexamethasone inhibition of rat hepatoma cell growth and cell cycle traverse is reversed by insulin.

(1) The growth of 7800 C1 Morris hepatoma cells was inhibited by dexamethasone. The inhibition was detectable at 1 nM and half-maximal effect was obtained with approx. 13 nM dexamethasone. About 80% growth inhibition was obtained with 250 nM of the hormone and the growth rate was normalized on cessation of treatment. (2) These hepatoma cells contain dexamethasone receptors with equilibrium dissociation constant of 0.24 nM and a capacity of 24 fmol/mg cell protein. Treatment of the cells with insulin did not change these dexamethasone binding properties. Binding experiments showed that 2, 10 and 100% of the receptors were occupied when the cells were incubated with 1 nM, 7 nM and 250 nM dexamethasone, respectively. (3) Insulin completely counteracted the growth inhibition by dexamethasone and antagonized the induction of peroxisomal acyl-CoA oxidase and tyrosine aminotransferase caused by the glucocorticoid. (4) Micro-flow fluorometry showed that the cultures had a major diploid DNA stem line and a minor tetraploid stem line. Changes in diploid, tetraploid and S phase cells of the diploid stem line were scored. Dexamethasone reduced the proportion of cells in S phase and of tetraploid cells. Insulin partly reversed the action of dexamethasone in S phase, but prevented the reduction in tetraploid cells caused by dexamethasone. (5) The mitotic rate was significantly reduced by dexamethasone and this effect was reversed by insulin. (6) Continuous [3H]methyl-thymidine labelling showed a growth fraction of unity in all treatment groups. (7) It is concluded that dexamethasone induces growth inhibition by reducing the G1-S transition. Insulin is able to counteract this effect and increase the rate of DNA synthesis.

Animals↗
PubMed1990

Insulin secretagogues induce Ca(2+)-like changes in cytoplasmic Mg2+ in pancreatic beta-cells.

The effects of insulin secretagogues on the cytoplasmic Mg2+ concentration ([Mg2+]i) of pancreatic beta-cells were studied in suspensions and in individual beta-cells using dual-wavelength fluorometry and the indicator mag-fura-2. Average [Mg2+]i was in the 800-900 microM range in a medium containing 3 mM glucose. When the sugar concentration was raised to 20 mM, the cells reacted with an initial lowering of [Mg2+]i followed by an increase. The sugar apparently also stimulated leakage of the Mg2+ indicator. Addition of 100 microM tolbutamide or raising the K+ concentration by 25 mM caused relatively rapid increases of [Mg2+]i. Methoxyverapamil prevented the [Mg2+]i-increasing actions of glucose, K+ and tolbutamide. The greatest change in [Mg2+]i was obtained when beta-cells were exposed to 100 microM carbachol. In this case there was a more than 10% lowering, which was reversed upon removal of the agonist. Measurements of [Mg2+]i are important not only for understanding fluctuations of this ion, but may also aid to elucidate the mechanisms involved in the regulation of cytoplasmic Ca2+.

Animals↗
PubMed1990

Non-NMDA receptor mediates cytoplasmic Ca2+ elevation in cultured hippocampal neurones.

Microscopic fluorometry of cultured rat hippocampal neurones revealed that the intracellular concentration of Ca2+ ([Ca2+]i) rises in response to the activation of excitatory amino acid receptor (EAA-R), which included not only N-methyl-D-aspartate subspecies but also kainate and quisqualate subspecies of EAA-R. Each EAA-R mediated [Ca2+]i rise consisted of the components dependent on and independent of the activity of the voltage-dependent Ca2+ channel. The receptor-mediated voltage-independent [Ca2+]i rise may be related to the modulation of synaptic transmission efficacy.

Animals↗
PubMed1994

5-HT1A receptor-mediated inhibition of N-type calcium current in acutely isolated ventromedial hypothalamic neuronal cells.

The effects of serotonin (5-hydroxytryptamine: 5-HT) and 5-HT1A agonist on voltage-gated calcium channels (VGCCs) in acutely isolated ventromedial hypothalamic (VMH) neuronal cells were studied using whole-cell configuration of the patch-clamp technique. 5-HT at 10 microM inhibited inward calcium current reversibly in 80% of cells. This inhibition was specific to N-type current. Because pindolol blocked the effect of 5-HT and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) mimicked the effect of 5-HT, the inhibitory effect of 5-HT appeared to be mediated via the 5-HT1A receptor. In the fura-2 fluorometry method, 8-OH-DPAT attenuated the [Ca2+]i increase induced by the depolarization stimulus of 50 mM K+. These results indicate that 5-HT suppresses Ca2+ entry through N-type channels in the VMH neurons via the 5-HT1A receptor and that the stimulating effect of 8-OH-DPAT on feeding behavior may be mediated by the blocking of Ca2+ entry through N-type channels.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1996

Long-term depression in rat visual cortex is associated with a lower rise of postsynaptic calcium than long-term potentiation.

To test the hypothesis that an input-associated rise of Ca2+ at postsynaptic sites beyond a certain threshold leads to the induction of long-term potentiation (LTP) while a lower rise below the threshold leads to long-term depression (LTD), the method of microscopic Ca2+ fluorometry was employed simultaneously with recordings of synaptic activity from layer II/III of visual cortical slices prepared from young rats. The conventional Ca2+ indicators, such as fura-2 or fluo-3, may interfere with intracellular processes for the induction of LTP/LTD because of their strong Ca(2+)-chelating action. To minimize such a problem, another Ca2+ indicator, rhod-2, was used since it has a much weaker Ca(2+)-chelating action than those indicators. In 16 slices loaded with rhod-2 through the perfusion medium, tetanic stimulation of theta-burst type was applied to layer IV of the cortex and changes in Ca2+ concentration were analyzed in layer II/III from which field potentials to test stimulation of layer IV were recorded simultaneously. In 7 slices in which weak tetanic stimulation consisting of 0.1 ms duration pulses was applied to layer IV, LTD of field responses was induced, while LTP was induced in 6 of the 9 slices in which strong tetanus consisting of 0.2 ms pulses was applied. In the 6 slices in which LTP was induced, the peak rise of fluorescence intensity during tetanus was 13.9 +/- 0.2 (S.E.M.) %, which was significantly (t-test, P < 0.01) higher than that (10.4 +/- 0.3%) in the 9 slices in which LTD was induced. In another series of experiments, rhod-2 was injected directly into 12 pyramidal cell-like neurons in layer II/III through patch pipettes, and changes in Ca2+ concentration in apical dendritic areas during tetanus were measured simultaneously with recordings of excitatory postsynaptic potentials (EPSPs) evoked by test stimulation of layer IV. It was found that LTP of EPSPs was induced in 4 cells which exhibited a strong rise of dendritic Ca2+ signal (197.1 +/- 18.5%) while LTD was induced in other 5 cells which showed a weak rise of the signal (31.0 +/- 4.1%). These results seem consistent with the above-mentioned, Ca(2+)-switching hypothesis for the induction of LTP and LTD in visual cortex.

Animals↗