Cortical Membranes Research Articles

MTORC2 regulates renal tubule sodium uptake by promoting ENaC activity.

The epithelial Na+ channel (ENaC) is essential for Na+ homeostasis, and dysregulation of this channel underlies many forms of hypertension. Recent studies suggest that mTOR regulates phosphorylation and activation of serum/glucocorticoid regulated kinase 1 (SGK1), which is known to inhibit ENaC internalization and degradation; however, it is not clear whether mTOR contributes to the regulation of renal tubule ion transport. Here, we evaluated the effect of selective mTOR inhibitors on kidney tubule Na+ and K+ transport in WT and Sgk1-/- mice, as well as in isolated collecting tubules. We found that 2 structurally distinct competitive inhibitors (PP242 and AZD8055), both of which prevent all mTOR-dependent phosphorylation, including that of SGK1, caused substantial natriuresis, but not kaliuresis, in WT mice, which indicates that mTOR preferentially influences ENaC function. PP242 also substantially inhibited Na+ currents in isolated perfused cortical collecting tubules. Accordingly, patch clamp studies on cortical tubule apical membranes revealed that mTOR inhibition markedly reduces ENaC activity, but does not alter activity of K+ inwardly rectifying channels (ROMK channels). Together, these results demonstrate that mTOR regulates kidney tubule ion handling and suggest that mTOR regulates Na+ homeostasis through SGK1-dependent modulation of ENaC activity.

Normal aging in rats and pathological aging in human Alzheimer’s disease decrease FAAH activity: Modulation by cannabinoid agonists

Anandamide is an endocannabinoid involved in several physiological functions including neuroprotection. Anandamide is synthesized on demand and its endogenous level is regulated through its degradation, where fatty acid amide hydrolase plays a major role. The aim of this study was to characterize anandamide breakdown in physiological and pathological aging and its regulation by CB1 and CB2 receptor agonists.Fatty acid amide hydrolase activity was analyzed in an independent cohort of human cortical membrane samples from control and Alzheimer’s disease patients, and in membrane and synaptosomes from adult and aged rat cerebral cortex. Our results demonstrate that fatty acid amide hydrolase activity decreases in the frontal cortex from human patients with Alzheimer’s disease and this effect is mimicked by Aβ1-40 peptide. This activity increases and decreases in aged rat cerebrocortical membranes and synaptosomes, respectively. Also, while the presence of JWH-133, a CB2 selective agonist, slightly increases anandamide hydrolysis in human controls, it decreases this activity in adults and aged rat cerebrocortical membranes and synaptosomes. In the presence of WIN55,212-2, a mixed CB1/CB2 agonist, anandamide hydrolysis increases in Alzheimer’s disease patients but decreases in human controls as well as in adult and aged rat cerebrocortical membranes and synaptosomes. Although a similar profile is observed in fatty acid amide hydrolase activity between aged rat synaptic endings and human Alzheimer’s disease brains, it is differently modulated by CB1/CB2 agonists. This modulation leads to a reduced availability of anandamide in Alzheimer’s disease and to an increased availability of this endocannabinoid in aging.

A search for functional histamine H4 receptors in the human, guinea pig and mouse brain.

Histamine H4 receptors are expressed in immune cells, but their potential role in the brain is less clear. Although H4 transcripts have been identified in human and rat brain, the presence of H4 receptors on the protein level has so far not been proven since appropriate antibodies fulfilling the strict criteria for G protein-coupled receptors are missing. Here, we searched for functional H4 receptors in human, guinea pig and mouse cortex. We studied whether H4 receptor activation is associated with increased GTPγS binding and reduced noradrenaline release. The latter two effects have been previously shown for H3 receptors, which, like the H4 receptors, are coupled to G i/o protein. G protein activation was studied using (35)S-GTPγS binding in cortical membranes. The electrically induced (3)H-noradrenaline release was determined in superfused cortical slices. The H4 agonist 4-methylhistamine failed to affect (35)S-GTPγS binding and/or noradrenaline release in human, guinea pig and mouse cortex although an H 3 receptor-mediated increase in (35)S-GTPγS binding and inhibition of noradrenaline release occurred in parallel experiments. In conclusion, functional H4 receptors increasing (35)S-GTPγS binding and/or decreasing noradrenaline release are not found in human, guinea pig and mouse cortex.

Apoptotic cells subjected to cold/warming exposure disorganize apoptotic microtubule network and undergo secondary necrosis.

Apoptotic microtubule network (AMN) is organized during apoptosis, forming a cortical structure beneath the plasma membrane which plays a critical role in preserving cell morphology and plasma membrane integrity. The aim of this study was to examine the effect of cold/warming exposure on apoptotic microtubules and plasma membrane integrity during the execution phase of apoptosis. We demonstrated in camptothecin-induced apoptotic H460 cells that cold/warming exposure disorganized apoptotic microtubules and allowed the access of active caspases to the cellular cortex and the cleavage of essential proteins in the preservation of plasma membrane permeability. Cleavage of cellular cortex and plasma membrane proteins, such as α-spectrin, paxilin, focal adhesion kinase and calcium ATPase pump (PMCA-4) involved in cell calcium extrusion resulted in increased plasma permeability and calcium overload leading apoptotic cells to secondary necrosis. The essential role of caspase-mediated cleavage in this process was demonstrated because the addition of the pan-caspase inhibitor z-VAD during cold/warming exposure that induces AMN depolymerization avoided the cleavage of cortical and plasma membrane proteins and prevented apoptotic cells to undergo secondary necrosis. Likewise, apoptotic microtubules stabilization by taxol during cold/warming exposure also prevented cellular cortex and plasma membrane protein cleavage and secondary necrosis. Furthermore, microtubules stabilization or caspase inhibition during cold/warming exposure was also critical for proper phosphatidylserine externalization and apoptotic cell clearance by macrophages. These results indicate that cold/warming exposure of apoptotic cells induces secondary necrosis which can be prevented by both, microtubule stabilization or caspase inhibition.

Positive allosteric modulation of the GHB high-affinity binding site by the GABAA receptor modulator monastrol and the flavonoid catechin

γ-Hydroxybutyric acid (GHB) is a metabolite of γ-aminobutyric acid (GABA) and a proposed neurotransmitter in the mammalian brain. We recently identified α4βδ GABAA receptors as possible high-affinity GHB targets. GABAA receptors are highly sensitive to allosteric modulation. Thus to investigate whether GHB high-affinity binding sites are also sensitive to allosteric modulation, we screened both known GABAA receptor ligands and a library of natural compounds in the rat cortical membrane GHB specific high-affinity [3H]NCS-382 binding assay. Two hits were identified: Monastrol, a positive allosteric modulator of GABA function at δ-containing GABAA receptors, and the naturally occurring flavonoid catechin. These compounds increased [3H]NCS-382 binding to 185–272% in high micromolar concentrations. Monastrol and (+)-catechin significantly reduced [3H]NCS-382 dissociation rates and induced conformational changes in the binding site, demonstrating a positive allosteric modulation of radioligand binding. Surprisingly, binding of [3H]GHB and the GHB high-affinity site-specific radioligands [125I]BnOPh-GHB and [3H]HOCPCA was either decreased or only weakly increased, indicating that the observed modulation was critically probe-dependent. Both monastrol and (+)-catechin were agonists at recombinant α4β3δ receptors expressed in Xenopus laevis oocytes. When monastrol and GHB were co-applied no changes were seen compared to the individual responses. In summary, we have identified the compounds monastrol and catechin as the first allosteric modulators of GHB high-affinity binding sites. Despite their relatively weak affinity, these compounds may aid in further characterization of the GHB high-affinity sites that are likely to represent certain GABAA receptors.

AB0993 Indirect Assessment of Amyloid A Deposits in Different Structures of the Kidneys – A Postmortem Clinicopathologic Study of 161 Rheumatoid Arthritis Patients

BackgroundAmyloidosis is a progressive, cumulative process, involving in its early stage only a few structures in some organs, and increasingly more in the later stages of the disease [1].ObjectivesThe aim...

Structure of the Small Dictyostelium discoideum Myosin Light Chain MlcB Provides Insights into MyoB IQ Motif Recognition

Dictyostelium discoideum MyoB is a class I myosin involved in the formation and retraction of membrane projections, cortical tension generation, membrane recycling, and phagosome maturation. The MyoB-specific, single-lobe EF-hand light chain MlcB binds the sole IQ motif of MyoB with submicromolar affinity in the absence and presence of Ca(2+). However, the structural features of this novel myosin light chain and its interaction with its cognate IQ motif remain uncharacterized. Here, we describe the NMR-derived solution structure of apoMlcB, which displays a globular four-helix bundle. Helix 1 adopts a unique orientation when compared with the apo states of the EF-hand calcium-binding proteins calmodulin, S100B, and calbindin D9k. NMR-based chemical shift perturbation mapping identified a hydrophobic MyoB IQ binding surface that involves amino acid residues in helices I and IV and the functional N-terminal Ca(2+) binding loop, a site that appears to be maintained when MlcB adopts the holo state. Complementary mutagenesis and binding studies indicated that residues Ile-701, Phe-705, and Trp-708 of the MyoB IQ motif are critical for recognition of MlcB, which together allowed the generation of a structural model of the apoMlcB-MyoB IQ complex. We conclude that the mode of IQ motif recognition by the novel single-lobe MlcB differs considerably from that of stereotypical bilobal light chains such as calmodulin.

The Drosophila MAST kinase Drop out is required to initiate membrane compartmentalisation during cellularisation and regulates dynein-based transport

Cellularisation of the Drosophila syncytial blastoderm embryo into the polarised blastoderm epithelium provides an excellent model with which to determine how cortical plasma membrane asymmetry is generated during development. Many components of the molecular machinery driving cellularisation have been identified, but cell signalling events acting at the onset of membrane asymmetry are poorly understood. Here we show that mutations in drop out (dop) disturb the segregation of membrane cortical compartments and the clustering of E-cadherin into basal adherens junctions in early cellularisation. dop is required for normal furrow formation and controls the tight localisation of furrow canal proteins and the formation of F-actin foci at the incipient furrows. We show that dop encodes the single Drosophila homologue of microtubule-associated Ser/Thr (MAST) kinases. dop interacts genetically with components of the dynein/dynactin complex and promotes dynein-dependent transport in the embryo. Loss of dop function reduces phosphorylation of Dynein intermediate chain, suggesting that dop is involved in regulating cytoplasmic dynein activity through direct or indirect mechanisms. These data suggest that Dop impinges upon the initiation of furrow formation through developmental regulation of cytoplasmic dynein.

Excitability and responsiveness of rat barrel cortex neurons in the presence and absence of spontaneous synaptic activity in vivo.

The amplitude and temporal dynamics of spontaneous synaptic activity in the cerebral cortex vary as a function of brain states. To directly assess the impact of different ongoing synaptic activities on neocortical function, we performed in vivo intracellular recordings from barrel cortex neurons in rats under two pharmacological conditions generating either oscillatory or tonic synaptic drive. Cortical neurons membrane excitability and firing responses were compared, in the same neurons, before and after complete suppression of background synaptic drive following systemic injection of a high dose of anaesthetic. Compared to the oscillatory state, the tonic pattern resulted in a more depolarized and less fluctuating membrane potential (Vm), a lower input resistance (Rm) and steeper relations of firing frequency versus injected current (F-I). Whatever their temporal dynamics, suppression of background synaptic activities increased mean Vm, without affecting Rm, and induced a rightward shift of F-I curves. Both types of synaptic drive generated a high variability in current-induced firing rate and patterns in cortical neurons, which was much reduced after removal of spontaneous activity. These findings suggest that oscillatory and tonic synaptic patterns differentially facilitate the input-output function of cortical neurons but result in a similar moment-to-moment variability in spike responses to incoming depolarizing inputs.

The Drosophila MAST kinase Drop out is required to initiate membrane compartmentalisation during cellularisation and regulates dynein-based transport.

Cellularisation of the Drosophila syncytial blastoderm embryo into the polarised blastoderm epithelium provides an excellent model with which to determine how cortical plasma membrane asymmetry is generated during development. Many components of the molecular machinery driving cellularisation have been identified, but cell signalling events acting at the onset of membrane asymmetry are poorly understood. Here we show that mutations in drop out (dop) disturb the segregation of membrane cortical compartments and the clustering of E-cadherin into basal adherens junctions in early cellularisation. dop is required for normal furrow formation and controls the tight localisation of furrow canal proteins and the formation of F-actin foci at the incipient furrows. We show that dop encodes the single Drosophila homologue of microtubule-associated Ser/Thr (MAST) kinases. dop interacts genetically with components of the dynein/dynactin complex and promotes dynein-dependent transport in the embryo. Loss of dop function reduces phosphorylation of Dynein intermediate chain, suggesting that dop is involved in regulating cytoplasmic dynein activity through direct or indirect mechanisms. These data suggest that Dop impinges upon the initiation of furrow formation through developmental regulation of cytoplasmic dynein.

Commentary: Morris-Coole/Epilepsia prize.

Commentary: Morris-Coole/Epilepsia prize.

Synthesis and evaluation of candidate PET radioligands for corticotropin-releasing factor type-1 receptors.

A radioligand for measuring the density of corticotropin-releasing factor subtype-1 receptors (CRF1 receptors) in living animal and human brain with positron emission tomography (PET) would be a useful tool for neuropsychiatric investigations and the development of drugs intended to interact with this target. This study was aimed at discovery of such a radioligand from a group of CRF1 receptor ligands based on a core 3-(phenylamino)-pyrazin-2(1H)-one scaffold. CRF1 receptor ligands were selected for development as possible PET radioligands based on their binding potency at CRF1 receptors (displacement of [(125)I]CRF from rat cortical membranes), measured lipophilicity, autoradiographic binding profile in rat and rhesus monkey brain sections, rat biodistribution, and suitability for radiolabeling with carbon-11 or fluorine-18. Two identified candidates (BMS-721313 and BMS-732098) were labeled with fluorine-18. A third candidate (BMS-709460) was labeled with carbon-11 and all three radioligands were evaluated in PET experiments in rhesus monkey. CRF1 receptor density (Bmax) was assessed in rhesus brain cortical and cerebellum membranes with the CRF1 receptor ligand, [(3)H]BMS-728300. The three ligands selected for development showed high binding affinity (IC50 values, 0.3-8nM) at CRF1 receptors and moderate lipophilicity (LogD, 2.8-4.4). [(3)H]BMS-728300 and the two (18)F-labeled ligands showed region-specific binding in rat and rhesus monkey brain autoradiography, namely higher binding density in the frontal and limbic cortex, and cerebellum than in thalamus and brainstem. CRF1 receptor Bmax in rhesus brain was found to be 50-120 fmol/mg protein across cortical regions and cerebellum. PET experiments in rhesus monkey showed that the radioligands [(18)F]BMS-721313, [(18)F]BMS-732098 and [(11)C]BMS-709460 gave acceptably high brain radioactivity uptake but no indication of the specific binding as seen in vitro. Candidate CRF1 receptor PET radioligands were identified but none proved to be effective for imaging monkey brain CRF1 receptors. Higher affinity radioligands are likely required for successful PET imaging of CRF1 receptors.

Impaired cortical mitochondrial function following TBI precedes behavioral changes

Traumatic brain injury (TBI) pathophysiology can be attributed to either the immediate, primary physical injury, or the delayed, secondary injury which begins minutes to hours after the initial injury and can persist for several months or longer. Because these secondary cascades are delayed and last for a significant time period post-TBI, they are primary research targets for new therapeutics. To investigate changes in mitochondrial function after a brain injury, both the cortical impact site and ipsilateral hippocampus of adult male rats 7 and 17 days after a controlled cortical impact (CCI) injury were examined. State 3, state 4, and uncoupler-stimulated rates of oxygen consumption, respiratory control ratios (RCRs) were measured and membrane potential quantified, and all were significantly decreased in 7 day post-TBI cortical mitochondria. By contrast, hippocampal mitochondria at 7 days showed only non-significant decreases in rates of oxygen consumption and membrane potential. NADH oxidase activities measured in disrupted mitochondria were normal in both injured cortex and hippocampus at 7 days post-CCI. Respiratory and phosphorylation capacities at 17 days post-CCI were comparable to naïve animals for both cortical and hippocampus mitochondria. However, unlike oxidative phosphorylation, membrane potential of mitochondria in the cortical lining of the impact site did not recover at 17 days, suggesting that while diminished cortical membrane potential at 17 days does not adversely affect mitochondrial capacity to synthesize ATP, it may negatively impact other membrane potential-sensitive mitochondrial functions. Memory status, as assessed by a passive avoidance paradigm, was not significantly impaired until 17 days after injury. These results indicate pronounced disturbances in cortical mitochondrial function 7 days after CCI which precede the behavioral impairment observed at 17 days.

CD44 Regulation of Endothelial Cell Proliferation and Apoptosis via Modulation of CD31 and VE-cadherin Expression

CD44 has been implicated in a diverse array of cell behaviors and in a diverse range of signaling pathway activations under physiological and pathophysiological conditions. We have documented a role for CD44 in mediating vascular barrier integrity via regulation of PECAM-1 (CD31) expression. We now report our findings on the roles of CD44 in modulating proliferation and apoptosis of microvascular endothelial cells via its modulation of CD31 and VE-cadherin expression and the Hippo pathway. In this report, we demonstrate persistent increased proliferation and reduced activations of both effector and initiator caspases in high cell density, postconfluent CD44 knock-out (CD44KO), and CD31KO cultures. We found that reconstitution with murine CD44 or CD31 restored the proliferative and caspase activation rates to WT levels. Moreover, we have confirmed that the CD31 ecto-domain plays a key role in specific caspase cascades as well as cell adhesion-mediated cell growth and found that CD31 deficiency results in a reduction in VE-cadherin expression. Last, we have shown that both CD44KO and CD31KO endothelial cells exhibit a reduced VE-cadherin expression correlating with increased survivin expression and YAP nuclear localization, consistent with inactivation of the Hippo pathway, resulting in increased proliferation and decreased apoptosis. These findings support the concept that CD44 mediates several of its effects on endothelia through modulation of adhesion protein expression, which, in addition to its known modulation of junctional integrity, matrix metalloproteinase levels and activation, interactions with cortical membrane proteins, and selected signaling pathways, plays a key role as a critical regulator of vascular function.

Lipid–protein interactions in plasma membranes of fiber cells isolated from the human eye lens

The protein content in human lens membranes is extremely high, increases with age, and is higher in the nucleus as compared with the cortex, which should strongly affect the organization and properties of the lipid bilayer portion of intact membranes. To assess these effects, the intact cortical and nuclear fiber cell plasma membranes isolated from human lenses from 41- to 60-year-old donors were studied using electron paramagnetic resonance spin-labeling methods. Results were compared with those obtained for lens lipid membranes prepared from total lipid extracts from human eyes of the same age group [Mainali, L., Raguz, M., O’Brien, W. J., and Subczynski, W. K. (2013) Biochim. Biophys. Acta]. Differences were considered to be mainly due to the effect of membrane proteins. The lipid-bilayer portions of intact membranes were significantly less fluid than lipid bilayers of lens lipid membranes, prepared without proteins. The intact membranes were found to contain three distinct lipid environments termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain. However, the cholesterol bilayer domain, which was detected in cortical and nuclear lens lipid membranes, was not detected in intact membranes. The relative amounts of bulk and trapped lipids were evaluated. The amount of lipids in domains uniquely formed due to the presence of membrane proteins was greater in nuclear membranes than in cortical membranes. Thus, it is evident that the rigidity of nuclear membranes is greater than that of cortical membranes. Also the permeability coefficients for oxygen measured in domains of nuclear membranes were significantly lower than appropriate coefficients measured in cortical membranes. Relationships between the organization of lipids into lipid domains in fiber cells plasma membranes and the organization of membrane proteins are discussed.

Functional activation of Gαq via serotonin2A (5-HT2A) and muscarinic acetylcholine M1 receptors assessed by guanosine-5׳-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding/immunoprecipitation in rat brain membranes

Functional coupling between serotonin2A (5-HT2A) receptors and Gαq proteins in native brain membranes has been sparsely reported thus far. In the present study, the guanosine-5׳-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding assay combined with immunoprecipitation using magnetic beads (Dynabeads Protein A) coated with anti-Gαq antibody was developed. Under experimental conditions optimised for assay constituents (GDP, MgCl2, and NaCl), for contents of membrane protein, anti-Gαq antibody, and Dynabeads Protein A, and for the incubation period, 5-HT stimulated specific [35S]GTPγS binding to Gαq in rat cerebral cortical membranes in a concentration-dependent and saturable manner, with a signal/noise ratio that was sufficiently high for further detailed pharmacological characterisation. This characterisation revealed an involvement of 5-HT2A receptors. Activation of Gαq proteins was also detectable by the addition of carbachol via muscarinic acetylcholine M1 receptors, (-)-epinephrine, and dopamine, but not by L-glutamate or (±)-baclofen. When 5-HT2A receptors and M1 receptors were stimulated simultaneously, there were non-additive effects, indicating that the two receptors were coupled to the same components of Gαq proteins in the rat cerebral cortex. This method will serve as an efficacious strategy for neurobiological investigations aimed at elucidating the physiological and pathological implications of signal transduction systems mediated via Gαq proteins coupled with 5-HT2A receptors and muscarinic acetylcholine M1 receptors.

Modes of direct modulation by taurine of the glutamate NMDA receptor in rat cortex

Taurine is an endogenous brain substance with robust neuromodulatory and possible neuroprotective properties. Though other mechanisms of action have been reported, its interaction with the NMDA (N-methyl-d-aspartic acid) receptor is undocumented. We investigated taurine׳s interaction with the NMDA receptor using electrophysiological and receptor binding approaches. The effects of taurine on field potential responses in layer-5 of prelimbic cortex in rat brain slices evoked by single-pulse electrical stimulation of ventral medial cortex were determined. Picrotoxin (80µM) was present in all control and drug solutions to block the Cl− channels associated with the GABA-, taurine-, and strychnine sensitive glycine- receptors. A typical response consisted of an NBQX (2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo-[f]-quinoxaline-7-sulfonamide)-sensitive negative wave (N1) followed by a positive wave (P1) and a broad negativity (N2), both sensitive to dl-AP5 (dl-2-amino-5-phosphonopentanoic acid) inhibition. Taurine exerted a 41.5±8.3% (n=9) voltage reduction within the late phase of N2. This taurine action was prevented by 100µM AP5, but not by 10µM nifedipine, supporting a direct modulation of NMDA receptor function by taurine, without requiring the involvement of the l-type Ca2+ channel. Taurine did not alter specific [3H] MK-801 binding to rat cortical membranes in the presence of glycine or glutamate; but inhibited spermine-potentiated specific [3H] MK-801 binding to NMDA receptors by 15–20% in the presence of glycine. In addition, taurine reduced the apparent affinity of the NMDA receptor for glycine (in the presence of spermine) by 10-fold. These results show that taurine interacts directly with the NMDA receptor by multiple mechanisms.

Phospholipid-Cholesterol Bilayers, Cholesterol Bilayer Domains, and Cholesterol Crystals were Detected in Lipid Dispersion Prepared from Lipids Extracted from Lens Nucleus of Old Human Donors

Human lens lipid membranes prepared using a rapid solvent exchange method from the total lipids extracted from the clear lens cortex and nucleus of 61- to 70-year-old donors were investigated. The measured cholesterol-to-phospholipid (Chol/PL) molar ratio in these preparations was extremely high, showing values of 1.8 and 4.4 for cortex and nucleus, respectively. We expect that at this elevated Chol content, the entire membrane will become saturated with Chol (as in the case of the cortical membrane) and the excess of Chol will form Chol crystals, presumably outside the membrane (as in the case of the nuclear membrane). Properties and organization of the lipid bilayer were investigated using electron paramagnetic resonance spin-labeling methods. Formation of Chol crystals was confirmed using the differential scanning calorimetry. We showed that in the lipid dispersion prepared from nuclear lipids Chol exists in three distinguished environments: (1) Chol dispersed in PL bilayer, (2) Chol in non-crystalline membrane domains (cholesterol bilayer domains, CBDs), and (3) Chol in crystals. In cortical membranes, because of the lower Chol content, Chol crystals were not detected. Amounts of Chol in CBDs were almost the same in cortical and nuclear membranes which indicates that Chol content in both membranes is close or exceeds the Chol solubility thresholds in these membranes. Profiles of cortical and nuclear membrane properties (alkyl-chain order, fluidity, oxygen transport parameter, and hydrophobicity) were very similar to each other and to those reported for cortical and nuclear lens lipid membranes of 41- to 60-year-old donors reported earlier. This confirms our earlier statement that saturation with Chol determines properties of the PL bilayer with the minor effect of the PL composition.

Optimum conditions of radioligand receptor binding assay of ligands of benzodiazepine receptors.

To obtain drugs which are more selective at benzodiazepine (BZD) receptors, design and synthesis of functionally selective ligands for BZD receptors is the current strategy of our pharmaceutical chemistry department. The affinity of newly synthesized ligands is assessed by radioligand receptor binding assays. Based on our previous studies, 2-phenyl-5-oxo-7-methyl-1,3, 4-oxadiazolo[a,2,3]-pyrimidine (compound A) was chosen for design and synthesis of new triazole derivatives as GABAA BZD receptor agonist. The cortical membrane of male Sprague-Dawley rats was prepared as the source of the BZD receptors. Different concentrations of membrane protein and [3H]-flumazenil were incubated at room temperature at different time periods to reach the steady-state. To saturate the receptors, increased amounts of radioligand were incubated with membrane protein. The bound and un-bound ligands were separated by centrifugation. The affinity of compound A was measured in competition studies at optimum conditions by displacement of [3H]-Flumazenil from rat cortical membrane. Based on results, the optimum conditions of radioligand receptor binding assay of benzodiazepines were 35 min incubation of ligands with 100 μg cortical membrane protein and 8.6 × 10-5 nmole 3H-flumazenil in a final volume of 0.5 mL Tris-HCl buffer (50 mM, pH 7.4) at 30 °C. The binding parameters of [3H]-flumazenil, Bmax and Kd were determined through saturation studies as 0.638 ± 0.099 pmol/mg and 1.35 ± 0.316 nM respectively. The affinity of compound A was 1.9 nM comparable with diazepam (1.53nM). This finding makes the compound an interesting lead for further optimization. Starting from this compound, new ligands were synthesized and screened in-vitro by competitive binding assays.

Significance of Arachnoid Dissection to Obtain Optimal Exposure of Lower Cranial Nerves and the Facial Nerve Root Exit Zone during Microvascular Decompression Surgery

This letter discusses the article2) entitled Delayed unilateral soft palate palsy without vocal cord involvement after microvascular decompression for hemifacial spasm recently published in J Korean Neurosurg Soc 53 : 364-367, 2013. In order to prevent cranial nerve palsies during microvascular decompression for hemifacial spasm, it is important to avoid retracting the cerebellum from the internal to the external side. Instead, it should be retracted from the lower to the upper side to obtain the optimal exposure of lower cranial nerves and the facial nerve root exit zone (FNREZ). The author of this manuscript devised an arachnoid dissection technique to expose the lower cranial nerves and the FNREZ. The retromastoid approach is most commonly conducted in the lateral decubitus position. The dura mater is incised along the sigmoid sinus, and the Cisterna magna is opened to drain the cerebrospinal fluid after careful retraction of the cerebellum from the lower to the upper side. The cerebellar cortical branches of the anterior inferior cerebellar artery (AICA), which is covered with an arachnoid membrane exterior to the lower cranial nerves, are exposed. Under the microscope, the outside of the arachnoid membrane is attached to the pia mater of the cerebellar cortical braches of AICA, which is dissected using an arachnoid knife and microscissors. At this point, the inner side of the arachnoid membrane of the cerebellar cortical braches of AICA should be preserved (Fig. 1). Next, the space between the cerebellar cortical braches of AICA and the cerebellar cortex is separated, and the sharp continuous arachnoid membrane dissection is extended towards the anterior and inferior end of lower cranial nerves modified by author's technique (Fig. 2)1). The cerebellar cortical branches of AICA are then retracted naturally towards the tentorium, and the choroid plexus, located in the lateral recess of the 4th ventricle, can be visualized. The offending artery and entire set of lower cranial nerves, facial nerves, including the FNREZ, can then be widely exposed (Fig. 3). Fig. 1 The outside of cortical anterior inferior cerebellar artery arachnoid membrane is dissected using microscissors. Fig. 2 The sharp coninuous arachnoid dissection is extended toward anterior and inferior end of lower cranial nerves. Fig. 3 The offending vessels and entire set of lower cranial nerves, facial nerve root exit zone are widely exposed. A : Left. B : Right. I think the significance of this arachnoid dissection procedures can prevent the lower cranial nerve palsies and also helpful to avoid cerebellar ischemia resulting from cerebellar cortical arterial compression/retraction during the operation.