Involvement of the 12-lipoxygenase pathway of arachidonic acid metabolism in homosynaptic long-term depression of the rat hippocampus

Low-frequency stimulation is associated with long-term depression (LTD) of synaptic efficacy in various brain structures. Like long-term potentiation (LTP), homosynaptic LTD in area CA1 of the hippocampus appears to require NMDA receptor activation, changes in postsynaptic calcium concentration and phospholipase A₂ (PLA₂) activation. Arachidonic acid (AA) is released after the activation of calcium-dependent phospholipases and free AA is rapidly metabolized to a family of bioactive products (the eicosanoids) which are thought to be both intracellular and extracellular messengers. In the present study, we investigated the involvement of the cyclooxygenase and lipoxygenase pathways of AA metabolism in the formation of homosynaptic LTD in the rat hippocampus. Stimulation at 1 Hz for 15 min was used to produce homosynaptic depression in area CA1 of hippocampal slices. LTD induction was partially blocked by bromophenacyl bromide (50–100 μM), a selective PLA₂ inhibitor, and by the a nonselective lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA; 100 μM). In contrast, the specific cyclooxygenase blocker indomethacin (100 μM) did not significantly reduce hippocampal LTD. Since NDGA interferes with LTD formation, we examined whether specific inhibitors of 5- and 12-lipoxygenases were capable of blocking LTD expression. The 12-lipoxygenase inhibitor baicalein at a concentration of 50 μM reduced LTP formation when given in the bath, an effect that was less pronounced with the 5-lipoxygenase inhibitor AA-861. These data suggest that the activation of endogenous PLA₂ and the formation of 12-lipoxygenase metabolites of AA may be important factors controlling the expression of hippocampal LTD.     

Structure and Behavior in Hydrophilic Matrix Sustained Release Dosage Forms: 4. Studies of Water Mobility and Diffusion Coefficients in the Gel Layer of HPMC Tablets Using NMR Imaging

Purpose. The purpose of this study was to characterise the water mobility in the gel layer of hydrating HPMC tablets. Water mobility in the gel layer of different HPMCs was studied. Methods. NMR imaging, a non-invasive technique, has been used to measure the spatial distribution of self-diffusion coefficient (SDC) and T₂ relaxation times across the gel layer. Results. It has been shown that there is a water mobility gradient across the gel layer of HPMC tablets. Although SDC and T₂ relaxation times in the outer parts of the gel layer approached that of free water, in the inner parts they decreased progressively. Water mobility and SDC in the gel layer of different HPMCs appeared to vary with degree of substitution of the polymer and the lowest values were obtained across the gel layer of K4M tablets. Conclusions. Water mobility varies across the gel layer of hydrating HPMC tablets and it is dependent on the degree of substitution of the polymer.     

Magnetization Transfer and T2 Relaxation Components in Tissue

T₂ relaxation makes an important contribution to tissue contrast in magnetic resonance (MR) imaging. Many tissues are known to exhibit multicomponent T₂ relaxation that suggests some compartmental segregation of mobile protons on a T₂ timescale. Magnetization transfer (MT) is another relaxation mechanism that can be used to produce tissue contrast in MR imaging. The MT process depends strongly on water-macromolecular interactions. To investigate the relationship between multicomponent T₂ relaxation and the MT process, multiecho T₂ measurements have been combined with MT measurements for freshly excised samples of cardiac muscle, striated muscle, and white matter. For muscle, short T₂ components show greater MT than long T₂ components, consistent with the belief that they represent distinct water environments. For white matter, quantitative MT measurements were identical for the two major T₂ components, apparently because of exchange between the T₂ compartments on a timescale characteristic of the MT experiment. Implications for accurate modeling of MT in tissue and the use of MT for MR image contrast are discussed.     

Magnetization transfer imaging of multiple sclerosis

While conventional magnetic resonance imaging (MRI) measures signal primarily from the hydrogen nuclei of water, magnetization transfer (MT) MRI indirectly detects macromolecular associated hydrogen nuclei via their magnetic interaction with the observable water. In the normal adult CNS, white matter exhibits the largest MT effect due to the macromolecular content of the highly structured and lipid rich myelin. Pathologies which alter the structural integrity and the relative macromolecular-water composition, such as multiple sclerosis (MS), therefore show abnormal MT. Conventional MRI, which has a high MS lesion detection sensitivity but poor specificity in terms of differentiating the pathological state of a plaque, can thus be supplemented by MT to provide more specific information on the extent of demyelination and axonal loss. In this paper we review the basic concepts of MT imaging and its role in MS lesion characterization.Financial support was provided by the Medical Research Council of Canada, Fonds de la Recherche en Santé du Québec, and the Killam Foundation.     

NMR Microscopy of Single Neurons Using Spin Echo and Line Narrowed 2DFT Imaging

NMR microimages of single neural cells were acquired at 500 MHz using a conventional spin echo pulse sequence and a line-narrowing sequence that eliminates susceptibility effects. The data show that any contribution to the measured T₂ relaxation rate arising from diffusion in local field inhomogeneities using spin echo sequences at high fields and high spatial resolution is relatively small. We conclude that the measured T₂ difference between the nucleus and cytoplasm in these cells represents primarily a true T₂ relaxation effect arising from the interactions of water with macromolecules in the two compartments and does not result from microsusceptibility differences. These observations have implications regarding water compartmentation in single cells and the interpretation of the MR characteristics of tissues in vivo.     

Concentration-dependent metabolism of diazepam in mouse liver

Previous mouse liver studies with diazepam (DZ),N-desmethyldiazepam (NZ), and temazepam (TZ) confirmed that under first-order conditions, DZ formed NZ and TZ in parallel. Oxazepam (OZ) was generatedvia NZ and not TZ despite that preformed NZ and TZ were both capable of forming OZ. In the present studies, the concentration-dependent sequential metabolism of DZ was studied in perfused mouse livers and microsomes, with the aim of distinguishing the relative importance of NZ and TZ as precusors of OZ. In microsomal studies, theK _ms andV _maxs, corrected for binding to microsomal proteins, were 34 μM and 3.6 nmole/min per mg and 239 μM and 18 nmole/min per mg, respectively, forN-demthylation andC ₃-hydroxylation of DZ. TheK _ms andV _maxs forN-demethylation andC ₃-hydroxylation of TZ and NZ, respectively, to form OZ, were 58 μM and 2.5 nmole/min per mg and 311 μM and 2 nmole/min per mg, respectively. The constants suggest that at low DZ concentrations, NZ formation predominates and is a major source of OZ, whereas at higher DZ concentrations, TZ is the important source of OZ. In livers perfused with DZ at input concentrations of 13 to 35 μM, the extraction ratio of DZ (E{DZ}) decreased from 0.83 to 0.60. NZ was the major metabolite formed although its appearance was less than proportionate with increasing DZ input concentration. By contrast, the formation of TZ increased disporportionately with increasing DZ concentration, whereas that for OZ decreased and paralleled the behavior of NZ. Computer simulations based on a tubular flow model and thein vitro enzymatic parameters provided a poorin vitro-organ correlation. TheE{DZ}, appearance rates of the metabolites, and the extraction ratio of formed NZ (E{NZ, DZ}) were poorly predicted; TZ was incorrectly identified as the major precursor of OZ. Simulations with optimized parameters imporved the correlations and identified NZ as the major contributor of OZ. Saturation of DZN-demethylation at higher DZ concentrations increased the role of TZ in the formation of OZ. The poor aqueous solubility (limiting the concentration range of substrates usedin vitro), avid tissue binding and the coupling of enzymatic reactions in liver, favoring sequential metabolism, are possible explanations for the poorin vitro-organ correlation. This work emphasizes the complexity of the hepatic intracellular milieu for drug metabolism and the need for additional modeling efforts to adequately describe metabolite kinetics. This work was supported by the Medical Research Council of Canada (MA-9104).     

Quantitative MRI of the brain in children with sickle cell disease reveals abnormalities unseen by conventional MRI

Conventional MRI (cMRI) has shown that brain abnormalities without clinical stroke can manifest in patients with sickle cell disease (SCD). We used quantitative MRI (qMRI) and psychometric testing to determine whether brain abnormalities can also be present in patients with SCD who appear normal on cMRI. Patients 4 years of age and older with no clinical evidence of stroke were stratified by cMRI as normal (n = 17) or abnormal (n = 13). Spin-lattice relaxation time (T1) of gray and white matter structures was measured by the precise and accurate inversion recovery (PAIR) qMRI method. Patient cognitive ability was assessed with a standard psychometric instrument (WISC-III or WISC-R). In all 30 patients with SCD, qMRI T1 was lower than in 24 age- and race-matched controls, in cortical gray matter (P < .0006) and caudate (P < .0009), as well as in the ratio of gray-to-white matter T1 (P < .008). In the 17 patients who were shown to be normal by cMRI, qMRI T1 was still lower than in controls, in both cortical gray matter (P < .02) and caudate (P < .004). Histograms of voxel T1 show that the proportion of voxels with T1 values intermediate between gray and white matter (ie, consistent with encephalomalacia) was 9% higher than controls in patients shown to be normal by cMRI (P < .05) and 15% higher than controls in patients shown to be abnormal by cMRI (P < .0005). The full scale intelligence quotient (FSIQ) of all patients with SCD was 75, compared to the FSIQ of 88 in a historical control group of patient siblings (P < .001). The FSIQ of patients shown to be normal by cMRI was 79, significantly lower than the FSIQ of patient siblings (P < .04). The FSIQ of 71 in patients shown to be abnormal by cMRI was significantly lower than both the patient siblings (P < .005) and the patients shown to be normal by cMRI (P < .04). Patients shown to be abnormal by cMRI scored lower than patients shown to be normal by cMRI, specifically on the subtests of vocabulary (P = .003) and information (P = .03). Cognitive impairment is thus significant, even in patients with SCD who were shown to be normal by cMRI, suggesting that cMRI may be insensitive to subtle neurologic damage that can be detected by qMRI. Because cognitive impairment can occur in children normal by cMRI, our findings imply that prophylactic therapy may be needed earlier in the course of SCD to mitigate neurologic damage.     

Effects of Polyethyleneglycol Chain Length and Phospholipid Acyl Chain Composition on the Interaction of Polyethyleneglycol-phospholipid Conjugates with Phospholipid: Implications in Liposomal Drug Delivery

Purpose. The purpose of this study was to investigate polyethyleneglycol(PEG)-phosphatidylethanolamine(PE) conjugate interaction with phospholipid bilayers, in an attempt to explain the dependence of liposome circulation time on formulation. Methods. Differential scanning calorimetry, electron microscopy, dynamic light scattering and NMR were the major methods used in the study. Results. Mixtures of PEG-phospholipid conjugates and phosphatidylcholine existed in three different physical states: a lamellar phase with components exhibiting some miscibility, a lamellar phase with components phase separated, and mixed micelles. Beyond 7 mol% of PEG(l,000–3,000)-dipalmitoyl phosphatidylethanolamine (DPPE), and 11 mol% PEG(5,000)-DPPE in dipalmitoyl phosphatidylcholine (DPPC), a strong tendency towards mixed micelle formation was observed. All concentrations of PEG(12,000)-DPPE and PEG(5,000)-DPPE beyond 8 mol% formed phase separated lamellae with phosphatidylcholine. Decreasing the acyl chain length from C_16:0 to C_14:0 caused a decrease in tendency towards micelle formation and phase separation. These tendencies increased upon increasing acyl chain length to C_18:0. Phase separation was at least partly due to PEG chain-chain interaction. This was supported by an increased fraction of PEG chains exhibiting a fast NMR transverse relaxation in DPPC/PEG(5,000)-DPPE mixtures as compared to that in distearoyl phosphatidylcholine (DSPC)/PEG(5,000)-dioleoyl-PE (DOPE). Conclusions. These phenomena are discussed in relation to both bilayer and steric stabilization of liposomes, and the lack of prolonged circulation with certain formulations is discussed.     

Pertussis toxin and N-ethylmaleimide inhibit histamine- but not calcium ionophore-induced endothelium-dependent relaxation

Summary Endothelium-dependent relaxation of the guinea pig pulmonary artery induced by histamine was inhibited by preincubation of the tissue with 10 M N-ethylmaleimide (NEM) for 10 min, whereas the endothelium-dependent relaxation induced by the calcium ionophore A 23187 was not affected by NEM. Pretreatment of the preparations with 0.2–1 g/ml pertussis toxin for 120 min inhibited concentration-dependently the histamine-induced relaxation. In contrast, endothelium-dependent relaxation in response to the calcium ionophore A 23187 was not affected by pertussis toxin. Since NEM and pertussis toxin are thought to interfere with membrane located GTP binding proteins, it is suggested that such a coupling protein is involved in the signal transduction of the histamine receptor leading to endothelium-dependent relaxation.A preliminary report of these results was presented at the autumn Meeting of the Deutsche Pharmakologische Gesellschaft, 1986 Send offprint requests to G. Weinheimer at the above address