A novel biomembrane model for electrochemical studies : characterisation and applications

This thesis describes the development and characterisation of a novel biomembrane model suitable for electrochemical investigation, and its applicability in studies of biologically related phenomena. The model system consists of a lipid monolayer, deposited at a polarisable liquid–liquid interface with the Langmuir–Blodgett technique. Combinations of ac and dc electrochemical techniques with theoretical models are employed to obtain information on the phase transfer and membrane interactions of charged therapeutics at the monolayer-covered interface. The thesis comprises six publications and a thorough introduction to biological and model membranes. The first part of the thesis surveys background literature with relevance to the present work. Basic properties of biological membranes are discussed and existing biomembrane models reviewed. The presented literature serves as a reference point for the development and evaluation of the model system described in this thesis, and is frequently referred to in the discussion of the results. In the second part of the thesis, the essential results of the publications are summarised. Instead of an article-by-article approach, results from various publications are jointly presented under appropriate headings. The development, characterisation and applicability of the model system are discussed. In the last-mentioned section, emphasis lies on methods applicable to drug development and delivery. To this end, the usefulness of the model system in the construction of pH – potential diagrams, determination of drug membrane activity, and studies of drug transfer through polyelectrolyte multilayers, is investigated. Finally, in view of the obtained results, the future prospects of the system are assessed.reviewe

The emerging pharmacology of TRPM8 channels: Hidden therapeutic potential underneath a cold surface

14 p., 2 figures, 2 tables and references.Transient receptor potential melastatin 8 (TRPM8) is a non-selective cation channel activated by cold temperature and cooling agents. TRPM8 is expressed in a subpopulation of cold-sensitive sensory neurons, as well as in the male urogenital system. TRPM8 is markedly upregulated in prostate cancer and in other tumors such as breast adenocarcinoma and melanoma. Moreover, recent studies suggest the potential involvement of TRPM8 channels in the pathophysiology of cold nociception and cold allodynia. This has led to a strong interest in the pursuit of novel modulators of TRPM8 channels. This review highlights our current knowledge of TRPM8 pharmacology and modulation mechanisms, detailing structural features important for TRPM8 gating by different agonists, the mechanism of antagonism by different compounds and the potential relevance of TRPM8 for treatment of various pathological conditions.We also like to acknowledge funding from the Spanish MICINN projects BFU2007-61855 to F. Viana, CONSOLIDER-INGENIO 2010 CSD2007-0002 and from the Fundación Marcelino Botín to C. Belmonte.Peer reviewe

The Influence of Cold Temperature on Cellular Excitability of Hippocampal Networks

The hippocampus plays an important role in short term memory, learning and spatial navigation. A characteristic feature of the hippocampal region is its expression of different electrical population rhythms and activities during different brain states. Physiological fluctuations in brain temperature affect the activity patterns in hippocampus, but the underlying cellular mechanisms are poorly understood. In this work, we investigated the thermal modulation of hippocampal activity at the cellular network level. Primary cell cultures of mouse E17 hippocampus displayed robust network activation upon light cooling of the extracellular solution from baseline physiological temperatures. The activity generated was dependent on action potential firing and excitatory glutamatergic synaptic transmission. Involvement of thermosensitive channels from the transient receptor potential (TRP) family in network activation by temperature changes was ruled out, whereas pharmacological and immunochemical experiments strongly pointed towards the involvement of temperature-sensitive two-pore-domain potassium channels (K2P), TREK/TRAAK family. In hippocampal slices we could show an increase in evoked and spontaneous synaptic activity produced by mild cooling in the physiological range that was prevented by chloroform, a K2P channel opener. We propose that cold-induced closure of background TREK/TRAAK family channels increases the excitability of some hippocampal neurons, acting as a temperature-sensitive gate of network activation. Our findings in the hippocampus open the possibility that small temperature variations in the brain in vivo, associated with metabolism or blood flow oscillations, act as a switch mechanism of neuronal activity and determination of firing patterns through regulation of thermosensitive background potassium channel activity. © 2012 de la Peña et al.During the course of this work, AM was supported by the Academy of Finland (107866) and a Juan de la Cierva fellowship from the Spanish Ministry of Education and Science. The work was also supported by funds from the Generalitat Valenciana: GVPRE/2008/205 to AM, GVAP/007/10 to EP and GRISOLIA/ 2010/020 to RC; the Spanish Ministry of Science and Innovation: projects SAF2010-14990 to FV and BFU2008-04425 and CONSOLIDER-INGENIO 2010 CSD2007-00023 to CB.Peer Reviewe

Differential effect of synaptic blockers on cooling-evoked responses.

<p><i>A</i>–<i>D</i>, Time courses of whole-cell current at a holding potential of −60 mV in four hippocampal neurons in the absence and presence of the synaptic blockers <i>A</i>, CNQX; <i>B</i>, AP-V; <i>C</i>, Bicuculline; <i>D</i>, Baclofen. <i>E</i>–<i>G</i>, Summary histogram of the number of events during cooling in the absence and presence of <i>E</i>, CNQX (n = 5); <i>F</i>, AP-V (n = 4); and <i>G</i>, Baclofen (n = 4). Statistical significance in panels D–F was assessed with repeated-measures 1-way ANOVA in combination with Dunnett’s post-test with respect to the first cooling stimulus in control conditions, and indicated with: *p<0.05; **p<0.01. All recordings, except B, were obtained in LCS. Time scale shown in A, applies to all traces (A–D).</p

Hippocampal neurons fire action potentials in response to cooling.

<p><i>A–B</i>, Time course of membrane potential change of two hippocampal neurons recorded in whole-cell current-clamp mode at −60 mV showing the cooling-evoked firing of action potentials. Note how the response of the neuron in A is completely abolished in the presence of a cocktail of synaptic blockers (20 µM CNQX+50 µM AP-V+5 µM bicuculline), whereas the neuron in B continues firing. The vertical lines in some of the voltage traces correspond with a pulse protocol for determination of membrane resistance. <i>C–D</i>, Effect of temperature on electrophysiological properties in current-clamp mode in the presence of a cocktail of synaptic blockers <i>C</i>, Membrane resistance was obtained from the membrane potential change in response to a 25 pA pulse of 250-millisecond-duration. <i>D,</i> Action potentials were evoked at rheobase using depolarizing current pulses of 450-ms-duration at a membrane potential of −60 mV. The neuron shown in C–D was silent in the presence of synaptic inhibitors. All recordings in [LCS].</p

Moderate cooling evokes large current responses in hippocampal neurons.

<p><i>A</i>, Time course of whole-cell current at −60 mV in a hippocampal neuron subjected to a cooling ramp from 35°C. Insets show individual current events (marked with an arrow) at baseline temperature and during the initial cooling period on an expanded time scale. The arrowhead marks the occurrence and temperature threshold of the first cold-evoked event. <i>B</i>, Response of the same neuron to heating from 35°C to 39°C and subsequent cooling. Note the absence of response during heating or during cooling from a higher baseline value. <i>C</i>, Number of events during cooling ramp for the neuron shown in A quantified in 2-second bins. <i>D</i>, Mean event frequency, <i>E</i>, mean amplitude and <i>F</i>, mean area during basal and cooling conditions (n = 15). Statistical significance in panels D–F was assessed with Student’s paired <i>t</i>-test (*p<0.05; **p<0.01. <i>G</i>–<i>H</i>, Cumulative probability histogram of event amplitude (<i>G</i>) and event area (<i>H</i>) at 35°C <i>vs.</i> during cooling of the recording shown in A. Note the tendency of cooling to shift both event amplitude and area towards larger values. <i>I</i>–<i>J</i>, Time course of cell-attached action currents recorded in the same hippocampal neuron, during identical cooling and heating protocol. Note the very similar characteristics of threshold and pattern as recorded in the whole-cell configuration. All recordings in this figure were performed in LCS (see Methods).</p

Temperature sensitive TRP channels are not involved in generating the cooling-evoked responses.

<p><i>A</i>, Time course of whole-cell current at −60 mV in a hippocampal neuron during repetitive cooling ramps in the absence and presence of thermo-TRP agonists menthol (100 µM), AITC (20 µM) and capsaicin (1 µM). The time scale bar indicates the zero current level. <i>B</i>−<i>D,</i> Summary histograms showing the average effect of the agonists on the <i>B,</i> threshold, <i>C</i>, average frequency, and <i>D,</i> relative amplitude of the temperature-induced responses during the descending part of a cooling ramp (n = 5). <i>E</i>, Time course of whole-cell current at −60 mV in a hippocampal neuron during cooling ramps in the absence and presence of the thermo-TRP antagonist BCTC (10 µM); <i>F,</i> Summary histogram showing the effect of BCTC on the mean frequency, amplitude and area of the cooling-evoked responses, n = 3. <i>G</i>−<i>H</i>, 20 µM HC-030031, n = 4; <i>I</i>−<i>J,</i> 10 µM ruthenium red, n = 2. In panels F, H, J, parameters in the presence of antagonist and during washout are normalized to data prior to antagonist application. In the different panels, statistical significance was assessed with repeated-measures 1-way-ANOVA in combination with Tukey’s post test, and is indicated with *p<0.05 where applicable. All records shown were obtained in HCS except the neuron in panel A.</p

Sensitivity of cooling-evoked responses to arachidonic acid, riluzole, chloroform and nifedipine supports the involvement of TREK channels.

<p>Sensitivity of cooling-evoked responses to arachidonic acid, riluzole, chloroform and nifedipine supports the involvement of TREK channels.</p