Biomedical subjects

J S Tauskela

Publications and source records attributed to J S Tauskela.

13 recordsLinked to original sources

PubMed2001

Cross-tolerance to otherwise lethal N-methyl-D-aspartate and oxygen-glucose deprivation in preconditioned cortical cultures.

In vitro ischemic preconditioning induced by subjecting rat cortical cultures to nonlethal oxygen-glucose deprivation protects against a subsequent exposure to otherwise lethal oxygen-glucose deprivation. We provide evidence that attenuation of the postsynaptic N-methyl-D-aspartate (NMDA) receptor- and Ca(2+)-dependent neurotoxicity underlies oxygen-glucose deprivation tolerance. It is demonstrated that extended tolerance to otherwise lethal NMDA or oxygen-glucose deprivation can be induced by either of their sublethal forms of preconditioning. These four pathways are linked, since NMDA receptor blockade during preconditioning by oxygen-glucose deprivation eliminates tolerance. These results suggest that NMDA tolerance, induced by nonlethal activation of these receptors during oxygen-glucose deprivation preconditioning, underlies oxygen-glucose deprivation tolerance. Several neurotoxic downstream Ca(2+)-dependent signaling events specifically linked to NMDA receptor activation are attenuated during otherwise lethal oxygen-glucose deprivation in preconditioned cultures. Specifically, calpain activation, as well as degradation of microtubule-associated protein-2 and postsynaptic density-95, are attenuated 2 h following otherwise lethal NMDA treatment alone or oxygen-glucose deprivation in preconditioned cultures. Formation of microtubule-associated protein-2-labeled dendritic varicosities is also attenuated in preconditioned cultures within 1 h of lethal oxygen-glucose deprivation or NMDA application. Intracellular Ca(2+) levels, measured using the high- or low-affinity dyes Fluo-4 (K(d) approximately equal 345 nM) or Fluo-4FF (K(d) approximately equal 9.7 microM) respectively, are markedly attenuated during lethal oxygen-glucose deprivation in preconditioned cultures.Collectively, the results suggest the attenuation of the postsynaptic NMDA-mediated component of otherwise lethal oxygen-glucose deprivation through the suppression of Ca(2+)-dependent neurotoxic signaling, a mechanism that is initially induced by transient nonlethal activation of this receptor during ischemic preconditioning.

Aniline Compounds↗

PubMed2000

Evaluation of glutathione-sensitive fluorescent dyes in cortical culture.

The sensitivity of six fluorophores to glutathione (GSH) was evaluated in living rat cortical neuronal/glial mixed cultures during the first 23 days in vitro (DIV). Four of the dyes require glutathione-S-transferase (GST) to form a fluorescent conjugate, potentially conferring specificity for GSH: these included t-butoxycarbonyl-Leu-Met-7-amino-4-chloromethylcoumarin (CMAC), 7-amino-4-chloromethylcoumarin (CMAC-blue), monochlorobimane (MCB), and 5-chloromethylfluorescein diacetate (CMFDA). The final two dyes examined, 2,3-naphthalenedicarboxaldehyde (NDA) and o-phthaldehyde (OPD), do not require GST for adduct formation with GSH. To examine the specificity of the dyes for GSH, cultures grown less than 6 DIV were pretreated with diethyl maleate or DL-buthionine-(S, R)-sulfoximine to deplete endogenous GSH. This resulted in a substantial loss of staining by CMAC, CMAC-blue, and MCB and partial loss of staining by OPD, indicating specificity for GSH, while staining by CMFDA or NDA was not altered, indicating a lack of specificity for GSH. Neurons experienced a dramatic decline in GSH levels relative to astrocytes between 5-6 DIV, as shown by a loss of neuronal staining with CMAC, CMAC-blue and MCB. This decrease in staining was not due to a decrease in GST activity, as neurons stained with the GST-insensitive OPD also exhibited a decline in GSH-sensitive staining. Immunolabeling experiments demonstrated that CMAC staining co-localized with GFAP-positive astrocytes, but not with MAP-2-positive neurons, in 18 DIV cultures. Finally, CMAC was exploited as a specific morphological marker of astrocytes in cultures aged >5 DIV. CMAC staining was employed to monitor astrocyte proliferation and to resolve astrocytes in living mixed cultures co-loaded with the Ca(2+)-sensitive dye, calcium green 5N-AM. GLIA 30:329-341, 2000. Published 2000 Wiley-Liss, Inc.

Animals↗

PubMed1999

Evidence from cultured rat cortical neurons of differences in the mechanism of ischemic preconditioning of brain and heart.

Ca2+ influx and activation of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) during nonlethal ischemic preconditioning have been implicated in the protection of the heart against subsequent lethal ischemic injury. Thus, we determined if Ca2+ influx, PKC and MAPK also mediate ischemic preconditioning-induced protection in neurons. Preconditioning by exposure of E18 rat cortical cultures to 90 min of nonlethal oxygen-glucose deprivation (OGD) 24 h prior to 180-240 min of lethal OGD was neuroprotective. Exposure to nominally free Ca2+, or blockade of the alpha-amino-hydroxy-5-methyl-isoxazolepropionate (AMPA) receptor with CNQX did not eliminate protection. MAPK activity did not change and PKC activity decreased by 50% relative to normal baseline levels at 0 and 24 h following preconditioning. The sustained decrease in PKC activity was not due to a loss of enzyme as determined from immunoblots using pan and epsilon-, beta- and zeta-specific PKC antibodies. Neuroprotection was maintained with pharmacological inhibition of PKC activity by staurosporine, chelerythrine and calphostin C and MAPK activity by PD 98059 during preconditioning, indicating that activation of these enzymes during preconditioning was not necessary for protection. Therefore, in contrast to cardiac tissue, ischemic preconditioning of neurons does not require activation of PKC and MAP kinase, and protection is maintained with substantial removal of extracellular Ca2+ or blockade of the AMPA receptor.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1998

Selective coupling of mu-calpain activation with the NMDA receptor is independent of translocation and autolysis in primary cortical neurons.

Excessive mu-calpain activation has been linked to several cellular pathologies including excitotoxicity and ischemia. In erythrocytes and other non-central nervous system (CNS) cells, calpain activation is thought to occur following a Ca2+-induced translocation of inactive cytosolic enzyme to membranes and subsequent autolysis. In the present report, we show that transiently exposing primary rat cortical neurons to lethal (50 microM) N-methyl-D-aspartic acid (NMDA) caused protracted calpain activation, measured as increased spectrin hydrolysis, but this was independent of translocation or autolysis of the protease. An anti-mu-calpain antibody showed that calpain was largely membrane associated in cortical neurons, and, consequently, neither translocation nor autolysis of the protease was observed following ionomycin or lethal NMDA treatment. By contrast, in rat erythrocytes, calpain was largely cytosolic and underwent rapid translocation and autolysis in response to ionomycin. Calpain-mediated spectrin hydrolysis was specifically coupled to Ca2+ entry through the NMDA receptor because nonspecific Ca2+ influx via ionomycin or KCl-mediated depolarization failed to activate the enzyme. Thus, calpain appears selectively linked to glutamate receptors in cortical neurons and regulated by mechanisms distinct from that occurring in many non-CNS cells. The data suggest that intracellular signals coupled to the NMDA receptor are responsible for activating calpain already associated with cellular membranes in cortical cells.

Animals↗

PubMed1998

A regulated environmental perfusion system for the study of anoxic or hypoxic cultured neurons using microfluorescence imaging and electrophysiology.

We describe the design and performance of a newly developed regulated environmental perfusion system (REPS). This system allows study of the effects of anoxia or hypoxia in cultured cells at physiological temperature, without the use of oxygen-scavenging compounds or metabolic inhibitors. The REPS incorporates a "canoe-shaped" flow-through chamber with access from above to allow positioning of pipettes for patch-clamp, microinjection, rapid-application perfusion, or microprobes for monitoring physical parameters. The combination of laminar flow and complete washout of perfusate within the chamber, and the use of a gas-tight perfusate delivery system and pressurized reservoirs containing media with pre-stabilized oxygen tensions (pO2 values) allow rapid production of accurate perfusate pO2 within the chamber. Perfusate pO2 in the chamber declined monoexponentially with time constants of </= 20 s to stable, pre-determined levels of 0 or 2 kPa (15 Torr). Shielding the gas/liquid interface of the chamber with an argon curtain only minimally decreased time constants at flow rates >/= 2 ml/min. The perfusion chamber of the REPS is easily mounted on the stage of an inverted microscope, for use with fluorescence imaging or electrophysiological studies of cultured cells. In tests with cultured rat cortical neurons, intracellular calcium concentration increased exponentially to values exceeding 1 microM during 10 min of anoxic insult, and returned to baseline values within 1 min after restoring normoxia.

Animals↗

PubMed1997

Evaluation of multiple-quantum-filtered 23Na NMR in monitoring intracellular Na content in the isolated perfused rat heart in the absence of a chemical-shift reagent.

The feasibility of employing triple-quantum-filtered (TQF) or double-quantum-filtered (DQF) 23Na NMR spectra to monitor intracellular Na (Nain) content in isolated rat hearts perfused in the absence of a chemical-shift reagent (SR) was investigated. This necessitated characterization of the following: first, the pool of Nain represented by the intracellular TQF (TQFin) spectrum; second, the maximum extent to which altered transverse relaxation times affect TQFin spectral amplitudes; and finally, the situations for which the SR-free method can reliably be applied. The rates of increase in peak amplitudes of both intracellular TQF spectra, adjusted for changes in both fast (T2f) and slow (T2s) transverse relaxation times, and intracellular single-quantum (SQin) spectra were identical during no-flow ischemia, indicating that TQFin and SQin spectra represent the same Nain population. Addition of an Na/K ATPase inhibitor, ouabain (>/=500 microM), and no-flow ischemia induced similar rates of increase of Nain content. However, the Nain level for which the T2 values started to increase was lower for ischemic (<140% of preischemic values) than for ouabain-exposed (>165%) hearts, which is consistent with the known earlier onset of intracellular swelling in ischemic hearts. Exposure of hearts to hyperosmotic perfusate (200 mM sucrose) increased [Nain], due to a decreased cell volume and an unchanged Nain content, but caused a decrease in T2 values, a trend opposite to that observed with exposure of hearts to ouabain or ischemia. T2 values therefore consistently correlated only with cell volume, not with Nain content or concentration, indicating an important role for intracellular macromolecule concentration in modulating transverse relaxation behavior. The combined effect of ischemia-induced increases in T2 values and their inhomogeneous broadened forms was an approximately 6% overestimation of Nain content from amplitudes of SR-aided TQFin spectra, indicating negligible effect of transverse relaxation-dependent alterations on TQFin spectral amplitudes. Thus, Nain content may be reliably determined from SR-free TQF spectra when the contribution from extracellular Na does not appreciably vary, such as during constant pressure perfusion. Following complete reduction in perfusion pressure, both SR-free TQF and DQF spectra respond to increases in Nain content. However, SR-free DQF NMR provides an estimate of Nain content much closer to that provided by the SR-aided method, due to the appreciable decrease of the extracellular DQF signal resulting from destructive interference between second- and third-rank tensors.

Animals↗

PubMed1996

Evaluation of triple-quantum-filtered 23Na NMR in monitoring of Intracellular Na content in the perfused rat heart: comparison of intra- and extracellular transverse relaxation and spectral amplitudes.

Multiple-quantum filtered (MQF) NMR offers the possibility of monitoring intracellular (IC) Na content in the absence of shift reagents (SR), provided that (i) the contribution from IC Na to the MQF spectrum is substantial and responds to a change in IC Na content, and (ii) the amplitude of the extracellular (EC) MQF component remains constant during a change in IC Na content. The validity and basis for these conditions were examined in isolated perfused rat hearts using SR-aided and SR-free triple-quantum filtered (TQF) 23NaNMR. Despite a myocardial Na content that was only approximately 1/70 that of EC Na. IC Na contributed to over 25% of the total TQF spectrum acquired in the absence of SR. Transverse relaxation times (T2) were approximately twice as long for EC compared to IC Na, despite SR-induced relaxation of T2 for the former pool. However, the efficiency of generation of the TQF signal was similar for IC and EC Na, indicating that a much greater percentage of IC relative to EC Na exhibits TQ coherence. During constant perfusion with ouabain (0.2 mM for 25 min) or with a hypoxic and aglycemic solution (50 min), the amplitude of the IC TQF spectrum increased by approximately 330% and -280%, respectively. In contrast, the amplitude of the EC TQF spectra remained essentially constant for both interventions. The amplitude for IC Na increased approximately 250% relative to baseline during no-flow ischemia (60 min), whereas the amplitude of the EC TQF spectra decreased by approximately 33% before stabilizing. In SR-free experiments, the TQF spectral amplitude increased approximately 2-fold during the constant perfusion interventions, but did not change significantly during no-flow ischemia. These data suggest that the change in the TQF spectral amplitude during constant perfusion interventions is from IC Na, and that TQF techniques in the absence of SR may be useful in monitoring IC Na during these interventions. The fall in the amplitude of the EC TQF spectral amplitude during no-flow ischemia complicates the use of TQF techniques without SR during this intervention.

Animals↗

PubMed1996

New double-quantum filtering schemes.

New double-quantum filtering (DQF) schemes are theoretically developed by reformulating the equations describing the double-quantum (DQ) signal. The equations describing the second- and third-rank DQ signals are simplified by restricting the RF phases as required for DQF. The equations are then factorized into two terms representing the separate contribution to the DQ signal from the RF pulses involved in the preparation and evolution times. This allows analysis of the DQ signal of a particular DQF scheme separately for each of these times in a concise manner. By use of the reformulated equations, the conventional DQF scheme is shown to be only one of four possible DQF schemes. The three new DQF schemes offer some desirable properties over the conventional DQF scheme. In the conventional DQF scheme, the third-rank DQ signal declines rapidly to null as the flip angles of the creation and readout RF pulses deviate from 90 degrees to 54.7 degrees or 125.3 degrees. In addition, the second- and third-rank DQ signals in the conventional DQF scheme are opposite in their polarities, resulting in attenuation of the total DQ signal due to destructive interference between them. In one of three new DQF schemes, the DQ signal does not vanish at 54.7 degrees and 125.3 degrees, but varies smoothly with the same functional dependence on the RF flip angles as the second-rank DQ and triple-quantum signals. Furthermore, in two of the three new DQF schemes, the second- and third-rank DQ signals have the same polarity so that the total DQ signal may be enhanced through constructive interference between them. These features of new DQF schemes have been confirmed experimentally.

Gels↗

PubMed1996

Intracellular calcium dynamics and cellular energetics in ischemic NG108-15 cells studied by concurrent 31P/19F and 23Na double-quantum filtered NMR spectroscopy.

The role of voltage-sensitive Ca2+ channels in mediating Ca2+ influx during ischemia was investigated in NG108-15 cells, a neuronal cell line that does not express glutamate-sensitive receptor-mediated Ca2+ channels. Concurrent 31P/19F and 23Na double-quantum filtered (DQF) NMR spectra were used to monitor cellular energy status, intracellular [Ca2+] ([Ca2+]i), and intracellular Na+ content in cells loaded with the calcium indicator 1,2-bis-(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA) during ischemia and reperfusion. Cells loaded with 5FBAPTA were indistinguishable from unloaded cells except for small immediate decreases in levels of phosphocreatine (PCr) and ATP. Ischemia induced a steady decrease in intracellular pH and PCr and ATP levels, and a steady increase in intracellular Na+ content; however, a substantial increase in [Ca2+]i (about threefold) was seen only following marked impairment of cellular energy status, when PCr was undetectable and ATP content was reduced to 55% of control levels. A depolarization-induced increase in [Ca2+]i could be completely blocked by 1 microM nifedipine, whereas up to 20 microM nifedipine had no effect on the increase in [Ca2+]i seen during ischemia. These data demonstrate that voltage-gated Ca2+ channels do not mediate significant Ca2+ flux during ischemia in this cell line and suggest an important role for Ca2+i stores, the Na+/Ca2+ antiporter, or other processes linked to cellular energy status in the increase in cytosolic Ca2+ level during ischemia.

Biological Transport↗

PubMed1995

Detection of an extracellular contribution from a second-rank tensor to the double-quantum-filtered 23Na NMR spectrum in the isolated perfused rat heart.

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Animals↗

PubMed1993

Response of the 23Na-NMR double-quantum filtered signal to changes in Na+ ion concentration in model biological solutions and human erythrocytes.

Double quantum filtered (DQF) 23Na-NMR signals were evaluated as a function of [Na+] at constant temperature in two model systems (bovine serum albumin (BSA) and Ficoll 400) and in human red blood cells (RBCs). In model systems, the ratio of double quantum filtered to single quantum (SQ) signal intensities was independent of [Na+], even over a wide range of Na+/K+ ratios. Varying the DQF preparation time affected only the DQF signal intensity. In contrast, in human red blood cells (RBCs) the shape and phase of the DQF intracellular Na+ signal (Na+in) varied as a function of preparation time. Similar observations in cartilage [Eliav, U., Shinar, H. and Navon, G. (1992) J. Magn. Reson. 28, 223-229] have been attributed to the generation of a second- and a third-rank tensor by the DQF pulse sequence, resulting from Na+ ion ordering. By using a DQF sequence which isolates the second-rank tensor only, this component was found to originate from the intracellular Na+ ion pool in human RBCs, as well as from interactions of Na+ ions with the extracellular face of the plasma membrane. The residual quadrupolar splitting for the signal originating from the former environment was shown to be less than the SQ linewidth, explaining its absence in SQ spectra, and this was confirmed by two-dimensional DQF 23Na-NMR experiments. By isolating the contribution from the third-rank tensor exclusively, the ratio of DQF:SQ signal intensities for Na+in in human RBCs was shown to be constant over a 4-fold change in [Na+in] produced by addition of an ionophore (nystatin). This indicates that such changes in physiological state do not alter the efficiency of DQF signal generation in human RBCs.

Animals↗

PubMed1992

A 31P-NMR spin-lattice relaxation and 31P[1H] nuclear Overhauser effect study of sonicated small unilamellar phosphatidylcholine vesicles.

The motional properties of the inner and outer monolayer headgroups of egg phosphatidylcholine (PC) small unilamellar vesicles (SUV) were investigated by 31P-NMR temperature-dependent spin-lattice relaxation time constant (T1) and 31P[1H] nuclear Overhauser effect (NOE) analyses. Three different aspects of the dynamics of PC headgroups were investigated using the T1 analysis. First, differences in the dynamics of the headgroup region of both surfaces of the SUV were measured after application of a chemical shift reagent, PrCl3, to either the extra- or intravesicular volumes. Second, the ability of the T1 experiment to resolve the different motional states was evaluated in the absence of shift reagent. Third, comparison between correlation times obtained from a resonance frequency dependent 31P[1H] NOE analysis allowed a determination of the applicability of a simplified motional model to describe phosphorus dipolar relaxation. Temperature-dependent 31P-NMR T1 values obtained for the individual monolayers at 81.0 and 162.0 MHz were modelled assuming that phosphorus undergoes both a dipolar and an anisotropic chemical shielding relaxation mechanism, each being described by the same correlation time, tau. At 162.0 MHz, the position of the T1 minimum for the inner monolayer was 9 degrees higher than that of the outer region, indicating a higher level of motional restriction for the inner leaflet, in agreement with 31P[1H] NOE measurements. The 162.0 MHz T1 profile of the combined SUV monolayers exhibited a smooth minimum located at the midpoint of the monolayer minima positions, effectively masking the presence of the individual surfaces. 31P[1H] NOE results obtained at 32.3, 81.0 and 162.0 MHz did not agree with those predicted from a simple dipolar relaxation model. These results suggest a T1-temperature method can neither discriminate two or more closely related motional time scales in a heterogeneous environment (such as incorporation of protein into lipid bilayers) nor allow accurate determination of the correlation time at the position of the minimum when the dipolar relaxation rate makes a significant contribution to the overall rate.

Magnetic Resonance Spectroscopy↗

PubMed1992

The size dependence of cholate-dialyzed vesicles on phosphatidylcholine concentration.

The size dependence of vesicles prepared by dialysis of cholate from phosphatidylcholine (PC) dispersions has been investigated as a function of lipid concentration (at a constant applied lipid: detergent molar ratio of 0.7). Gel filtration of dialyzed samples produced a symmetrical profile shape, although quasielastic laser light scattering analysis of the fractions revealed an asymmetrical range of sizes about the peak for solutions containing elevated lipid concentrations. Vesicle diameters increased by approximately 20 nm for PC concentrations ranging from 10 to a maximum of 45 mg/ml. This was attributed to mixed micelle sizes being proportional to lipid concentration, since the diameters of vesicles produced from dialysis are determined by mixed micelle sizes. Before commencement of dialysis, mixed micelle sizes are proportional to lipid concentration and, although dialysis causes an increase in mixed micelle sizes, the phase ratios attained are larger for solutions containing elevated lipid concentrations.

Cholic Acid↗