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A P Koretsky

Publications and source records attributed to A P Koretsky.

At least 37 records · Page 2Linked to original sources
PubMed1998

Comparison of the effects of ORG 30029, dobutamine and high perfusate calcium on function and metabolism in rat heart.

Cardiac contractility may be enhanced via multiple cellular mechanisms resulting in varied effects on cardiac energetics. The mechanisms that account for the varied energetic responses are not well understood. The purpose of this investigation was to compare the effects of the calcium sensitizing agent ORG 30029 (N-hydroxy-5,6-dimethoxy-benzo[b]thiophene-2-carboximidamide hydrochloride, a calcium sensitizing agent which increases contractility without increasing calcium transients significantly), dobutamine and high perfusate calcium on contractility and energetics. Langendorff-perfused rat hearts were stimulated with ORG 30029, dobutamine and high perfusate calcium in graduated concentrations while myocardial oxygen consumption (MVO2) and force-time integral were measured. ORG 30029, dobutamine and high perfusate calcium increased contractility in a dose-dependent manner. Despite an increase of 50% in systolic pressure and a 17% increase in force-time integral from control, ORG 30029 had no significant effect on MVO2 at the lower concentrations (n = 6). However, dobutamine (n = 4) and high perfusate calcium (n = 4) caused a 65% increase in systolic pressure and a 17% increase in force-time integral and a 50% and 41% increase in MVO2 respectively (P < 0.05). High energy phosphates (by 31P NMR), and lactate production were unaltered by these agents, suggesting that metabolism was steady state. Basal metabolism tended to increase slightly with dobutamine but not with ORG 30029 or high perfusate calcium. ORG 30029, dobutamine, and high perfusate calcium increase contractility in perfused rat hearts with disparate effects on energetics. These differences may be accounted for, in part, by differences in energy expenditure for calcium handling.

Animals↗
PubMed1998

Radial echo-planar imaging.

A new ultrafast magnetic resonance imaging pulse sequence named radial echo-planar imaging (rEPI) is introduced. The sequence is based on a modification of the echo-planar imaging (EPI) sequence to scan k-space radially, in an attempt to combine the speed of EPI with the benefits of radial sampling. Like in EPI, all the desired lines in k-space are scanned consecutively in opposite directions. The unique feature of this new sequence, however, is that the orientation of the readout gradient is incrementally rotated, so that all the echoes are refocused through the center of k-space. Therefore, rEPI data are acquired in a polar grid, and image reconstruction can be done either by means of filtered back-projection or by regridding the data to a Cartesian matrix followed by 2D Fourier transform. First results show that rEPI images can be acquired with the same speed and signal-to-noise ratio of EPI images. rEPI images are also shown to be less sensitive to off-resonance effects than EPI images. Further studies are underway to investigate the usefulness of rEPI for spectroscopic imaging and applications affected by motion.

Echo-Planar Imaging↗
PubMed1998

Simultaneous glutamate and perfusion fMRI responses to regional brain stimulation.

Functional magnetic resonance imaging (fMRI) rests on the assumption that regional brain activity is closely coupled to regional cerebral blood flow (rCBF) in vivo. To test the degree of coupling, cortical brain activity was locally stimulated in rats by reversed microdialysis infusion of picrotoxinin, alphagamma-aminobutyric acid-A antagonist. Before and during the first 30 minutes of infusion, simultaneous fMRI (rCBF) and neurochemical (interstitial glutamate concentration) measures of brain activity were highly correlated (r = 0.83). After 30 minutes of picrotoxinin-induced stimulation, glutamate levels decreased but rCBF remained elevated, suggesting that additional factors modulate the relationship between neuronal neurotransmitters and hemodynamics at these later stages.

Animals↗
PubMed1998

Fluorescence measurement of calcium transients in perfused rabbit heart using rhod 2.

Surface fluorescence spectroscopy of the beating heart to measure cytosolic calcium has been limited by the need to use ultraviolet excitation light for many of the commonly used calcium indicators. Ultraviolet light in the heart produces a high level of background fluorescence and is highly absorbed, limiting tissue penetration. Visible wave-length fluorescence dyes such as rhod 2 are available; however, the lack of spectral shift with calcium binding precludes the use of ratio techniques to account for changes in cytosolic dye concentration. We have developed a method for in vivo quantitation of cytosolic rhod 2 concentration that in conjunction with calcium-dependent fluorescence measurements permits estimation of cytosolic calcium levels in perfused rabbit hearts. Reflective absorbance of excitation light by rhod 2 loaded into myocardium was used as an index of dye concentration and the ratio of fluorescence intensity to absorbance as a measure of cytosolic calcium concentration. Endothelial cell loading of rhod 2 was found to be minimal (< 5%), and dye leak rate out of the cytosol was slow, with approximately 5% loss of dye fluorescence occurring between 10 and 30 min after dye loading. Rhod 2 loading into subcellular compartments, determined by manganese quenching, was also minimal (< 5%). The dissociation constant of rhod 2 for calcium was measured in vitro to be 500 nM, and this value increased to 710 nM in the presence of 0.5 mM myoglobin. On the basis of this value and in vivo fluorescence measurements, cytosolic calcium concentration in the rabbit heart was found to be 229 +/- 90 nM at end diastole and 930 +/- 130 nM at peak systole, with peak fluorescence preceding peak ventricular pressure by approximately 40 ms. This technique should facilitate detailed analysis of calcium transients from the whole heart.

Animals↗
PubMed1998

Absence of myofibrillar creatine kinase and diaphragm isometric function during repetitive activation.

Creatine kinase (CK) provides ATP buffering in skeletal muscle and is expressed as 1) cytosolic myofibrillar CK (M-CK) and 2) sarcomeric mitochondrial CK (ScCKmit) isoforms that differ in their subcellular localization. We compared the isometric contractile and fatigue properties of 1) control CK-sufficient (Ctl), 2) M-CK-deficient (M-CK[-/-]), and 3) combined M-CK/ScCKmit-deficient null mutant (CK[-/-]) diaphragm (Dia) to determine the effect of the absence of M-CK activity on Dia performance in vitro. Baseline contractile properties were comparable across groups except for specific force, which was approximately 16% lower in CK[-/-] Dia compared with M-CK[-/-] and Ctl Dia. During repetitive activation (40 Hz, (1)/(3) duty cycle), force declined in all three groups. This decline was significantly greater in CK[-/-] Dia compared with Ctl and M-CK[-/-] Dia. The pattern of force decline did not differ between M-CK[-/-] and Ctl Dia. We conclude that Dia isometric muscle function is not absolutely dependent on the presence of M-CK, whereas the complete absence of CK acutely impairs isometric force generation during repetitive activation.

Adenylate Kinase↗
PubMed1997

The role of creatine kinase in inhibition of mitochondrial permeability transition.

Cyclosporin A sensitive swelling of mitochondria isolated from control mouse livers and from the livers of transgenic mice expressing human ubiquitous mitochondrial creatine kinase occurred in the presence of both 40 microM calcium and 5 microM atractyloside which was accompanied by a 2.5-fold increase over state 4 respiration rates. Creatine and cyclocreatine inhibited the latter only in transgenic liver mitochondria. Protein complexes isolated from detergent solubilised rat brain extracts, containing octameric mitochondrial creatine kinase, porin and the adenine nucleotide translocator, were reconstituted into malate loaded lipid vesicles. Dimerisation of creatine kinase in the complexes and exposure of the reconstituted complexes to >200 microM calcium induced a cyclosporin A sensitive malate release. No malate release occurred with complexes containing octameric creatine kinase under the same conditions.

Animals↗
PubMed1997

Functional equivalence of creatine kinase isoforms in mouse skeletal muscle.

Creatine kinase (CK) is a highly conserved enzyme abundant in skeletal muscle that has a key role in high energy phosphate metabolism. The localization of the muscle isoenzyme of CK (MM-CK) to the M line and the sarcoplasmic reticulum of myofibrils has been suggested to be important for proper force development in skeletal muscle. The importance of this subcellular compartmentation has not been directly tested in vivo. To test the role of myofibrilar localization of CK, the consequences of a complete CK isoform switch from MM-CK to the brain (BB-CK) isoform, which does not localize to the M line, was studied in transgenic mouse skeletal muscle. In MM-CK knockout mice there are large contractile defects. When MM-CK was replaced by BB-CK, the aberrant contractile phenotypes seen in MM-CK knockout mice were returned to normal despite the lack of myofibrillar localization. These results indicate that CK compartmentation to the myofibril of skeletal muscle is not essential for contractile function and that there is functional equivalence of creatine kinase isoforms in supporting cellular energy metabolism.

Animals↗
PubMed1997

Estimation of water extraction fractions in rat brain using magnetic resonance measurement of perfusion with arterial spin labeling.

The model used for calculating perfusion by MRI techniques that use endogenous water as a tracer assumes that arterial water is a freely diffusible tracer. Evidence shows that this assumption is not valid in the brain at high blood flow rates, at which movement of water into and out of the microvasculature becomes limited by diffusion across the blood-brain barrier. In this work, the arterial spin-labeling technique is used to show that fraction of arterial water that is dependent on blood flow rate remains in the vasculature and does not exchange with brain tissue water. By using perfusion measurements without and with magnetization transfer (MT) effects, one can distinguish arterial label that exchanges into tissue because blood has much smaller MT than brain tissue. Using this technique, the extraction fraction for water is measured in the rat brain at various cerebral blood flow rates. At high flow rates (approximately 5 ml/g/min), the extraction fraction for water is found to be about 45% in rat brain. Disruption of the blood-brain barrier with D-mannitol caused an increase in the extraction fraction for water. It was possible to form an image related to the extraction fraction for water. The ability to estimate the amount of vascular water exchanging with tissue water by MRI may represent a noninvasive approach to detect the integrity of the blood-brain barrier.

Animals↗
PubMed1997

Evidence for the exchange of arterial spin-labeled water with tissue water in rat brain from diffusion-sensitized measurements of perfusion.

The extraction fraction of vascular water in rat brain is investigated by means of diffusion measurements of arterial spin labeled water at varying cerebral blood flow (CBF) values. The apparent diffusion coefficient (ADC) of the difference of the proton magnetization signal in the brain acquired with and without continuous arterial spin labeling is modeled to provide a measure of the amount of arterial water in tissue and vasculature and thus of the extraction fraction. The tissue and vascular portion of the arterial spin labeled water are differentiated based on their diffusion characteristics in a manner analogous to the intravoxel incoherent motion (IVIM) method. The amount of labeled arterial water that exchanges with tissue water is determined by estimating the fraction of the total signal that is associated with the slow-decaying component of a biexponential fit to the normalized difference signal between the magnetization of brain tissue acquired with and without arterial spin labeling. The results indicate that, at normal CBF (1.15 +/- 0.21 ml x g(-1) x min[-1]), about 90% of the arterial spin labeled water diffuses with an ADC of (1.21 +/- 0.37) x 10[-3] mm2 s[-1]), which is equal to tissue. At high CBF, an increasing fraction of the labeling water has a fast-pseudo-diffusion coefficient due to a decrease in water extraction fractions. The results also show that the contribution of vascular water to the measurement of perfusion by techniques that use endogenous water as a tracer can be efficiently eliminated by the use of diffusion sensitizing gradients with small effective b values (b approximately 20 s/mm2), enabling these techniques to monitor true changes in tissue perfusion.

Animals↗
PubMed1997

Manganese ion enhances T1-weighted MRI during brain activation: an approach to direct imaging of brain function.

Present techniques for functional MRI rely on detecting changes in hemodynamics that result as a consequence of brain activation. It would be useful if MRI techniques could be developed that enable imaging of a parameter directly related to neuronal activity. Influx of calcium into neurons is necessary for release of neurotransmitters. Divalent manganese ions (Mn2+) can enter cells through voltage-gated calcium channels and Mn2+ is paramagnetic. Mn2+ accumulation in brain due to activation should alter relaxation times offering an approach to sensitize MRI to calcium influx in the brain. To test this idea, T1-weighted MRI was obtained from the rat brain in the presence of a continuous intravenous infusion of 3.6 mumol/min MnCl2. In the anesthetized rat brain, signal enhancement was detected in regions corresponding to ventricles. Activation of the brain with glutamate led to increase in MRI signal intensity in the brain to 238 +/- 23% of the original. This increase in signal was dependent on the presence of MnCl2 and was not due to changes in blood flow. It was necessary to break the blood brain barrier with mannitol to make Mn2+ accessible to the active sites for efficient detection. Enhancement of MRI signal in the brain was also detected with decreasing anesthesia and with somatosensory stimulation. Due to the slow clearance of Mn2+ from the stimulated region of the brain, MRI enhancement could also be detected after stimulation that occurred on awake, behaving rats outside the magnet. These data indicate that MnCl2 shows potential as a MRI contrast agent that is directly sensitive to brain activation.

Animals↗
PubMed1997

Near-simultaneous hemoglobin saturation and oxygen tension maps in mouse brain using an AOTF microscope.

A newly developed microscope using acousto-optic tunable filters (AOTFs) was used to generate in vivo hemoglobin saturation (SO2) and oxygen tension (PO2) maps in the cerebral cortex of mice. SO2 maps were generated from the spectral analysis of reflected absorbance images collected at different wavelengths, and PO2 maps were generated from the phosphorescence lifetimes of an injected palladium-porphyrin compound using a frequency-domain measurement. As the inspiratory O2 was stepped from hypoxia (10% O2), through normoxia (21% O2), to hyperoxia (60% O2), measured SO2 and PO2 levels rose accordingly and predictably throughout. A plot of SO2 versus PO2 in different arterial and venous regions of the pial vessels conformed to the sigmoidal shape of the oxygen-hemoglobin dissociation curve, providing further validation of the two mapping procedures. The study demonstrates the versatility of the AOTF microscope for in vivo physiologic investigation, allowing for the generation of nearly simultaneous SO2 and PO2 maps in the cerebral cortex, and the frequency-domain detection of phosphorescence lifetimes. This class of study opens up exciting new possibilities for investigating the dynamics of hemoglobin and O2 binding during functional activation of neuronal tissues.

Acoustics↗
PubMed1997

Expression of functional mitochondrial creatine kinase in liver of transgenic mice.

The mitochondrial isoform of creatine kinase (MiCK) is localized to the mitochondrial intermembrane space, and its precise role in vivo is still actively being investigated. Here, we report a transgenic mouse model in which MiCK is expressed in liver, a tissue that does not normally express significant levels of CK. Expression of the genomic clone for human, ubiquitous MiCK was controlled by the promoter/enhancer region of the transthyretin gene. Three of seven founder mice were chosen to establish lines and had MiCK activity values ranging from 13 to 269 micromol x min(-1) x g wet wt(-1). Differential centrifugation and histochemical staining demonstrated that >90% of the CK activity is localized to the mitochondrial intermembrane space. An unusual mitochondrial morphology characterized by an angular nature to the membranes was detected using electron microscopy in the transgenic line expressing the highest levels of MiCK. Increasing hepatic total creatine levels led to a return to normal mitochondrial morphology. 31P-nuclear magnetic resonance spectroscopy demonstrated that the expressed MiCK is capable of producing and utilizing phosphocreatine. These mice will be useful for investigating gain of function effects of MiCK in cellular energetics.

Animals↗
PubMed1997

Differential effects of creatine kinase isoenzymes and substrates on regeneration in livers of transgenic mice.

Creatine kinase (CK) has been implicated in affecting cell growth, and the CK substrates creatine (Cr) and cyclocreatine (CyCr) have been shown to have anti-tumor activity. The influence of Cr and CyCr on liver regeneration following major hepatectomy was evaluated in normal and transgenic mice expressing the human ubiquitous mitochondrial isoform of CK (CK-mit) or the brain isoform of CK (CK-B) or livers expressing both CK-mit and CK-B (CK-comb). Expression of CK isoenzymes had little effect on liver regeneration in the absence of dietary supplementation with Cr or CyCr as assayed by the increase in liver mass. Dietary supplementation with Cr and CyCr significantly reduced liver growth in normal mice. Liver regeneration was almost completely inhibited in mice expressing CK-mit in the presence of Cr. Livers expressing CK-mit regenerated better than normal livers in the presence of CyCr. In mice expressing CK-B, Cr and CyCr had opposite effects from those found in CK-mit mice. In the presence of CyCr, regeneration was inhibited in livers expressing CK-B, and, in the presence of Cr, CK-B-expressing livers regenerated better than normal livers. The amount of DNA synthesized 2 days after hepatectomy confirmed the results obtained from measurements of liver mass for all groups. Growth and DNA synthesis were completely abolished by Cr in CK-mit mice, whereas CyCr mainly affected growth 2 days after hepatectomy in CK-B-expressing mice. Coexpression of the CK isoforms in CK-comb mice ameliorated the effects detected with either isoform alone. Inhibition of growth by Cr and CyCr was not correlated to water accumulation. These results clearly demonstrate isoenzyme and substrate-specific effects of CK on cell growth.

Animals↗
PubMed1997

Combined myofibrillar and mitochondrial creatine kinase deficiency impairs mouse diaphragm isotonic function.

Creatine kinase (CK) is an enzyme central to cellular high-energy phosphate metabolism in muscle. To characterize the physiological role of CK in respiratory muscle during dynamic contractions, we compared the force-velocity relationships, power, and work output characteristics of the diaphragm (Dia) from mice with combined myofibrillar and sarcomeric mitochondrial CK deficiency (CK[-/-]) with CK-sufficient controls (Ctl). Maximum velocity of shortening was significantly lower in CK[-/-] Dia (14.1 +/- 0.9 Lo/s, where Lo is optimal fiber length) compared with Ctl Dia (17.5 +/- 1.1 Lo/s) (P < 0.01). Maximum power was obtained at 0.4-0.5 tetanic force in both groups; absolute maximum power (2,293 +/- 138 W/m2) and work (201 +/- 9 J/m2) were lower in CK[-/-] Dia compared with Ctl Dia (2,744 +/- 146 W/m2 and 284 +/- 26 J/m2, respectively) (P < 0.05). The ability of CK[-/-] Dia to sustain shortening during repetitive isotonic activation (75 Hz, 330-ms duration repeated each second at 0.4 tetanic force load) was markedly impaired, with CK[-/-] Dia power and work declining to zero by 37 +/- 4 s, compared with 61 +/- 5 s in Ctl Dia. We conclude that combined myofibrillar and sarcomeric mitochondrial CK deficiency profoundly impairs Dia power and work output, underscoring the functional importance of CK during dynamic contractions in skeletal muscle.

Adenosine Triphosphatases↗
PubMed1997

Dilated cardiomyopathy in transgenic mice with cardiac-specific overexpression of tumor necrosis factor-alpha.

The failing human heart expresses tumor necrosis factor-alpha (TNF-alpha). However, its pathophysiological significance is not clear. We previously reported that robust overexpression of TNF-alpha in the murine heart causes lethal myocarditis. In this study, we modified the transgene to reduce the production of TNF-alpha by preserving the destabilizing sequence in TNF-alpha cDNA. Expression was driven by the murine alpha-myosin heavy chain promoter. Use of this modified construct allowed to the establish a mutine transgenic line (TG). TG offspring were examined at 6, 12, and 24 weeks. All showed a significantly higher heart weight-to-body weight ratio. Northern blot analysis confirmed the expression of transgene in the heart, and enzyme-linked immunosorbent assay demonstrated the presence of TNF-alpha protein. The TG heart demonstrated a mild, diffuse, lymphohistiocytic interstitial inflammatory infiltrate. Cardiomyocyte necrosis and apoptosis were present but not abundant. Magnetic resonance imaging showed that the TG heart was significantly dilated with reduced ejection fraction. Although the left ventricular dP/dtmax was not different at baseline, its responsiveness to isoproterenol was significantly blunted in TG. Atrial natriuretic factor was expressed in the TG ventricle. A group of TG died spontaneously, and subsequent autopsies revealed exceptional dilation of the heart, increased lung weight, and pleural effusion, suggesting that they died of congestive heart failure. The cumulative mortality rate at 6 months was 23%. In conclusion, the mouse overexpressing TNF-alpha recapitulated the phenotype of congestive heart failure. This provides a novel model to elucidate the role of this cytokine in the development of congestive heart failure.

Animals↗
PubMed1996

Contractile and metabolic effects of increased creatine kinase activity in mouse skeletal muscle.

The effects of increased expression of creatine kinase (CK) in skeletal muscle were studied in control and transgenic animals homozygous for expression of the B subunit of CK. CK activity was 47% higher in transgenic gastrocnemius muscle. The CK activity was distributed as follows: 45 +/- 1% MM dinner, 31 +/- 4% MB dimer, and 22 +/- 5% BB dimer. No significant differences in metabolic or contractile proteins were detected except for a 22% decrease in lactate dehydrogenase activity and a 9% decrease in adenylate kinase activity. The only significant effect in contractile activity was that the rise time of a 5-s isometric contraction was 28% faster in the transgenic muscle. 31P nuclear magnetic resonance (NMR) spectra were obtained from control and transgenic muscles during mechanical activation, and there were no NMR measurable differences detected. These results indicate that a 50% increase in CK activity due to expression of the B subunit does not have large effects on skeletal muscle metabolism or contractile function. Therefore, control muscle has sufficient CK activity to keep up with changes in cellular high-energy phosphates except during the early phase of intense contractile activity.

Adaptation, Physiological↗
PubMed1995

Multi-slice MRI of rat brain perfusion during amphetamine stimulation using arterial spin labeling.

When a single coil is used to measure perfusion by arterial spin labeling, saturation of macromolecular protons occurs during the labeling period. Induced magnetization transfer contrast (MTC) effects decrease tissue water signal intensity, reducing the sensitivity of the technique. In addition, MTC effects must be properly accounted for in acquiring a control image. This forces the image to a single slice centered between the labeling plane and the control plane. In this work, a two-coil system is presented as a way to avoid saturation of macromolecular spins during arterial spin labeling. The system consists of one small surface coil for labeling the arterial water spins, and a head coil for MRI, actively decoupled from the labeling coil by using PIN diodes. It is shown that no signal loss occurs due to MTC effects when the two-coil system is used for MRI of rat brain perfusion, enabling three-dimensional perfusion imaging. Using the two-coil system, a multi-slice MRI sequence was used to study the regional effects of amphetamine on brain perfusion. Amphetamine causes significant increases in perfusion in many areas of the brain including the cortex, cingulate, and caudate putamen, in agreement with previous results using deoxyglucose uptake to monitor brain activation.

Amphetamine↗