High Prevalence Of Insulin Resistance Assessed By The Glucose Clamp Technique In Hormonal And Non-hormonal Contraceptive Users.

Objective To assess the prevalence of insulin resistance (IR) and associated factors in contraceptive users. Methods A total of 47 women 18 to 40 years of age with a body mass index (kg/m(2)) < 30, fasting glucose levels < 100 mg/dl and 2-hour glucose level < 140 mg/dl after a 75-g oral glucose load were submitted to a hyperinsulinemic-euglycemic clamp. The women were distributed in tertiles regarding M-values. The analysed variables were use of combined hormonal/non-hormonal contraception, duration of use, body composition, lipid profile, glucose levels and blood pressure. Results IR was detected in 19% of the participants. The women with low M-values presented significantly higher body fat mass, waist-to-hip ratio, fasting insulin, HOMA-IR and were nulligravida, showed > 1 year of contraceptive use and higher triglyceride levels. IR was more frequent among combined oral contraceptive users, however no association was observed after regression analysis. Conclusions The prevalence of IR was high among healthy women attending a family planning clinic independent of the contraceptive method used with possible long-term negative consequences regarding their metabolic and cardiovascular health. Although an association between hormonal contraception and IR could not be found this needs further research. Family planning professionals should be proactive counselling healthy women about the importance of healthy habits.

Elf magnetic field influence on ION Channels studied by Patch Clamp Technique: exposure set up and "Whole Cell" measurements on Potassium currents

The aim of this thesis was to study the effects of extremely low frequency (ELF) electromagnetic magnetic fields on potassium currents in neural cell lines ( Neuroblastoma SK-N-BE ), using the whole-cell Patch Clamp technique. Such technique is a sophisticated tool capable to investigate the electrophysiological activity at a single cell, and even at single channel level. The total potassium ion currents through the cell membrane was measured while exposing the cells to a combination of static (DC) and alternate (AC) magnetic fields according to the prediction of the so-called ‘ Ion Resonance Hypothesis ’. For this purpose we have designed and fabricated a magnetic field exposure system reaching a good compromise between magnetic field homogeneity and accessibility to the biological sample under the microscope. The magnetic field exposure system consists of three large orthogonal pairs of square coils surrounding the patch clamp set up and connected to the signal generation unit, able to generate different combinations of static and/or alternate magnetic fields. Such system was characterized in term of field distribution and uniformity through computation and direct field measurements. No statistically significant changes in the potassium ion currents through cell membrane were reveled when the cells were exposed to AC/DC magnetic field combination according to the afore mentioned ‘Ion Resonance Hypothesis’.

On the function of proton-gated ion channels ASICs and TRPV1 : a patch-clamp study

AbstractAcidosis is encountered during tissue inflammation and triggers pain in humans. H+-gated ion channels are expressed at high levels in sensory neurons of the peripheral nervous system. Ion channels from two different families present the required pH sensitivity to detect the acidosis associated with peripheral inflammation: Acid-Sensing Ion Channels (ASICs) and the Transient Receptor Potential Vanilloid-1 (TRPV1) channel.ASICs are members of the Degenerin/Epithelial Na+ Channel family of ion channels. Six ASIC subunits have been identified in mammals (ASICla, -lb, -2a, -2b, -3 and -4). ASICs form In-activated voltage-insensitive homo- or heterotrimeric Na+ channels. TRPV1 is a member of the TRP family of ion channels and forms non-selective cation channels that mediate a sustained current. TRPV1 is activated by H+, heat (T>43°C), lipids, capsaicin, voltage and other stimuli. A stimulus can increase TRPV1 response to a different stimulus. For example H+ can shift the capsaicin concentration dependence of TRPV1 to lower values. ASICs and TRPV1 have been shown to be involved in inflammatory pain. Using the patch-clamp technique, we studied different aspects of the function of ASICs and TRPV1 in the physiological context of pain.In the first part of this thesis, we characterize the effect of a temperature increase from 25 to 35°C on the function of ASICs and TRPV1 in transfected CHO cells and primary cultures of rat DRG sensory neurons. ASICs give rise to transient currents while TRPV1 mediates a sustained current. In addition, ASICs and TRPV1 respond to H+ with distinct pH dependences. We assess the relative contribution of ASICs and TRPV1 to H+-evoked electrical signaling in rat DRG neurons and we conclude that ASICs are the most important pH sensors in the pH range 7.4 to 6.0 at 35°C in sensory neurons.ASICs and TRPV1 are expressed in the epithelium lining the lumen of the bladder (urothelium). The Bladder Pain Syndrome/Interstitial Cystitis (BPS/IC) is a painful condition associated with a dysfunction of the urothelial barrier and with inflammation. In the second part of this thesis, we show that human urothelial cells -the cell line TEU2 and primary cultures of human bladder urothelium- express functional ASICs but no functional TRPV1 channels. In addition, we show that the levels of ASIC2 and ASIC3 mRNA are increased in the urothelium of patients suffering from BPS/IC. These data suggest that ASICs are involved in the pathology of BPS/IC.Finally, we demonstrate that APETx2 inhibits the sensory neuron specific voltage-dependent Na+ channel Nav1.8. APETx2 was previously shown to inhibit homo- or heterotrimeric ASIC3- containing channels with IC5o from 0.08 to 1 μΜ. We show that APETx2 also inhibits Nav1.8 with an ICsoof «2.6 μΜ. APETx2 reduces the maximal conductance and induces a depolarizing shift in the voltage dependence of activation of Nav1.8. In current-clamp experiments, APETx2 reduces the number of action potentials (APs) evoked by a current ramp. Nav1.8 mediates most of the current during the AP upstroke and has been shown to be an important mediator of inflammatory pain. The fact that APETx2 inhibits two ion channels involved in inflammatory pain suggests that APETx2 or derivatives may represent novel analgesic compounds.RésuméL'acidose tissulaire est observée durant l'inflammation et entraine la douleur chez l'humain. Des canaux ioniques activés par les protons (H+) sont fortement exprimés dans les neurones sensoriels du système nerveux périphérique. De ceux-ci, les Acid-Sensing Ion Channels [ASICs) et Transient Receptor Potential Vanilloid-1 (TRPV1) présentent une sensibilité adéquate à l'acidité pour servir de détecteurs d'acidose.Les ASICs sont membres de la famille Degenerin/Epithelial Na* Channel. Six sous-unités ASIC ont été identifiées chez les mammifères (ASICla, -lb, -2a, -2b, -3 et -4). Les ASICs forment des canaux sélectifs au Na\ insensibles au voltage et activés par les H+. Les canaux fonctionnels sont des homo- ou hétérotrimères de sous-unités ASIC. TRPV1 est un membre de la famille TRP de canaux ioniques. Les canaux TRPV1 sont activés par les H+, la chaleur (T>43°Ç), les lipides, la capsaicine, le voltage et d'autres stimulus. L'activation de TRPV1 entraine un courant soutenu non-sélectif. Un stimulus peut augmenter la réponse de TRPV1 à un autre stimulus. Les H+ peuvent, par exemple, induire un décalage vers des valeurs plus faibles de la courbe de dépendance à la concentration de TRPV1 pour la capsaicine. Il a été démontré que les ASICs et TRPV1 sont impliqués dans la douleur inflammatoire. En utilisant la technique du patch-clamp, nous avons étudié différents aspects de la fonction des ASICs et de TRPV1 dans des contextes associés à la douleur.Dans la première partie de cette thèse, nous caractérisons l'effet d'une augmentation de température de 25 à 35°C sur la fonction des canaux ASICs et TRPV1, dans des cellules CHO transfectées et dans des cultures primaires de neurones sensoriels (DRG) de rat. L'activation des ASICs entraine l'apparition d'un courant transitoire tandis que l'activation de TRPV1 entraine un courant soutenu. De plus, les ASICs et TRPV1 possèdent des dépendances au pH différentes. Nous évaluons la contribution relative des ASICs et de TRPV1 au signalement électrique induit par les H+ et nous concluons que les ASICs sont les senseurs d'acidité les plus importants dans les neurones sensoriels, dans le domaine de pH de 7.4 à 6.0, à température corporelle.Les ASICs et TRPV1 sont exprimés dans l'épithélium recouvrant l'intérieur de la vessie (l'urothélium). Le Bladder Pain Syndrome/Interstitial Cystitis (BPS/IC) est une condition médicale douloureuse associée à une dysfonction de la barrière urothéliale et à une inflammation. Dans la seconde partie de cette thèse, nous démontrons que des cellules urothéliales (de la lignée cellulaire TEU2) et des cellules provenant de cultures primaires d'épithéliums de vessies humaines expriment des canaux ASIC fonctionnels mais pas de TRPV1 fonctionnels. De plus, nous montrons que le niveau d'expression de ASIC2 et -3 est augmenté dans l'urothélium de la vessie de patients souffrant de BPS/IC. Ces données suggèrent que les ASICs sont impliqués dans la pathologie BPS/IC.Pour finir, nous démontrons que la toxine APETx2 inhibe le canal spécifique aux neurones sensoriels Nav1.8, un membre de la famille des canaux sodiques dépendants du potentiel. Il a été démontré précédemment que la toxine APETx2 inhibe les canaux contenant une ou plusieurs sous-unités ASIC3 avec un ICso entre 0.08 et 1 μΜ. Nous montrons que la toxine APETx2 inhibe Nav1.8 avec un IC50 de «2.6 μΜ. La toxine APETx2 réduit la conductance maximale et induit un décalage de la dépendance au potentiel de Nav1.8 vers des valeurs plus positives. Dans des expériences de courant imposé sur des neurones sensoriels, la toxine APETx2 réduit le nombre de potentiels d'action induits par une rampe de courant. Nav1.8 est responsable de la majeure partie du courant durant la phase ascendante du potentiel d'action et a été démontré comme étant un médiateur important de la douleur inflammatoire. L'inhibition de deux types de canaux, impliqués dans la douleurs inflammatoire, par la toxine APETx2, suggère que cette dernière ou ses dérivés représentent des composés analgésiques prometteurs.

Untersuchungen zur Zell-Transistor-Kopplung mittels der Voltage-Clamp-Technik

Untersuchungen zur Zell-Transistor Kopplung mittels der Voltage-Clamp TechnikIn der vorliegenden Arbeit wird die extrazelluläre Einkopplung elektrischer Signale von Zellen in Transistoren hinsichtlich der an der Kopplung beteiligten Parameter untersucht. Dafür werden Zellen aus Primärkulturen und von Zell-Linien direkt auf den aktiven Sensorflächen der hergestellten Chips kultiviert. Für die Experimente werden n- und p-Kanal Feldeffekttransistoren (FET) sowie Extended-Gate-Elektroden (EGE) mit Gold- und Titanoberflächen entwickelt.Zur Untersuchung der Kopplungseigenschaften werden die neuronale Zell-Linie SH-SY5Y, die humane Endothel Zell-Linie EA.hy-926 sowie als Primärzellen hippocampale Neuronen und Kardiomyozyten embryonaler und neonataler Ratten eingesetzt. Die Voltage-Clamp Technik erlaubt die Untersuchung spannungsgesteuerter Ionenkanäle in der Zellmembran. Maßgebend für den Signalverlauf des extrazellulär eingekoppelten Signals ist der Ionenstrom von Na+, K+ und Ca2+ durch die Membran im Kontaktbereich zwischen Zelle und Sensor.Die Kopplung kann elektrisch mithilfe eines Ersatzschaltkreises beschrieben werden, der alle beteiligten elektrischen Größen der Membran und der Ionenströme, sowie die Parameter des Kontaktbereichs und des Sensors enthält.Die Simulation der extrazellulären Signale zeigt, dass die beobachteten Signalformen nur durch eine Erhöhung der Ionenkanaldichten und dadurch einer deutlich vergrößerten Leitfähigkeit der Ionenarten im Kontaktbereich gegenüber der freien Membran erklärt werden können.

Force-controlled patch clamp of beating cardiac cells.

From its invention in the 1970s, the patch clamp technique is the gold standard in electrophysiology research and drug screening because it is the only tool enabling accurate investigation of voltage-gated ion channels, which are responsible for action potentials. Because of its key role in drug screening, innovation efforts are being made to reduce its complexity toward more automated systems. While some of these new approaches are being adopted in pharmaceutical companies, conventional patch-clamp remains unmatched in fundamental research due to its versatility. Here, we merged the patch clamp and atomic force microscope (AFM) techniques, thus equipping the patch-clamp with the sensitive AFM force control. This was possible using the FluidFM, a force-controlled nanopipette based on microchanneled AFM cantilevers. First, the compatibility of the system with patch-clamp electronics and its ability to record the activity of voltage-gated ion channels in whole-cell configuration was demonstrated with sodium (NaV1.5) channels. Second, we showed the feasibility of simultaneous recording of membrane current and force development during contraction of isolated cardiomyocytes. Force feedback allowed for a gentle and stable contact between AFM tip and cell membrane enabling serial patch clamping and injection without apparent cell damage.

Nuclear hourglass technique: An approach that detects electrically open nuclear pores in Xenopus laevis oocyte

Nuclear pore complexes (NPCs) mediate both active transport and passive diffusion across the nuclear envelope (NE). Determination of NE electrical conductance, however, has been confounded by the lack of an appropriate technical approach. The nuclear patch clamp technique is restricted to preparations with electrically closed NPCs, and microelectrode techniques fail to resolve the extremely low input resistance of large oocyte nuclei. To address the problem, we have developed an approach for measuring the NE electrical conductance of Xenopus laevis oocyte nuclei. The method uses a tapered glass tube, which narrows in its middle part to 2/3 of the diameter of the nucleus. The isolated nucleus is sucked into the narrow part of the capillary by gentle fluid movement, while the resulting change in electrical resistance is monitored. NE electrical conductance was unexpectedly large (7.9 ± 0.34 S/cm2). Evaluation of NPC density by atomic force microscopy showed that this conductance corresponded to 3.7 × 106 NPCs. In contrast to earlier conclusions drawn from nuclear patch clamp experiments, NPCs were in an electrically “open” state with a mean single NPC electrical conductance of 1.7 ± 0.07 nS. Enabling or blocking of active NPC transport (accomplished by the addition of cytosolic extracts or gp62-directed antibodies) revealed this large NPC conductance to be independent of the activation state of the transport machinery located in the center of NPCs. We conclude that peripheral channels, which are presumed to reside in the NPC subunits, establish a high ionic permeability that is virtually independent of the active protein transport mechanism.

Membrane transport mechanisms probed by capacitance measurements with megahertz voltage clamp.

We have used capacitance measurements with a 1-microsecond voltage clamp technique to probe electrogenic ion-transporter interactions in giant excised membrane patches. The hydrophobic ion dipicrylamine was used to test model predictions for a simple charge-moving reaction. The voltage and frequency dependencies of the apparent dipicrylamine-induced capacitance, monitored by 1-mV sinusoidal perturbations, correspond to single charges moving across 76% of the membrane field at a rate of 9500 s-1 at 0 mV. For the cardiac Na,K pump, the combined presence of cytoplasmic ATP and sodium induces an increase of apparent membrane capacitance which requires the presence of extracellular sodium. The dependencies of capacitance changes on frequency, voltage, ATP, and sodium verify that phosphorylation enables a slow, 300- to 900-s-1, pump transition (the E1-E2 conformational change), which in turn enables fast, electrogenic, extracellular sodium binding reactions. For the GAT1 (gamma-aminobutyric acid,Na,Cl) cotransporter, expressed in Xenopus oocyte membrane, we find that chloride binding from the cytoplasmic side, and probably sodium binding from the extracellular side, results in a decrease of membrane capacitance monitored with 1- to 50-kHz perturbation frequencies. Evidently, ion binding by the GAT1 transporter suppresses an intrinsic fast charge movement which may originate from a mobility of charged residues of the transporter binding sites. The results demonstrate that fast capacitance measurements can provide new insight into electrogenic processes closely associated with ion binding by membrane transporters.

Nouveaux indices de suppression de la lipolyse par l'insuline déterminés lors de l'hyperglycémie provoquée par voie orale : comparaisons avec le clamp euglycémique-hyperinsulinémique et les paramètres métaboliques chez les femmes

Résumé : Une dysrégulation de la lipolyse des tissus adipeux peut conduire à une surexposition des tissus non-adipeux aux acides gras non-estérifiés (AGNE), qui peut mener à un certain degré de lipotoxicité dans ces tissus. La lipotoxicité constitue, par ailleurs, l’une des causes majeures du développement de la résistance à l’insuline et du diabète de type 2. En plus de ses fonctions glucorégulatrices, l’insuline a pour fonction d’inhiber la lipolyse et donc de diminuer les niveaux d’AGNE en circulation, prévenant ainsi la lipotoxicité. Il n’y a pas d’étalon d’or pour mesurer la sensibilité de la lipolyse à l’insuline. Le clamp euglycémique hyperinsulinémique constitue la méthode étalon d’or pour évaluer la sensibilité du glucose à l’insuline mais il est aussi utilisé pour mesurer la suppression de la lipolyse par l’insuline. Par contre, cette méthode est couteuse et laborieuse, et ne peut pas s’appliquer à de grandes populations. Il existe aussi des indices pour estimer la fonction antilipolytique de l’insuline dérivés de l’hyperglycémie provoquée par voie orale (HGPO), un test moins dispendieux et plus simple à effectuer à grande échelle. Cette étude vise donc à : 1) Étudier la relation entre les indices de suppressibilité des AGNE par l’insuline dérivés du clamp et ceux dérivés de l’HGPO; et 2) Déterminer laquelle de ces mesures corrèle le mieux avec les facteurs connus comme étant reliés à la dysfonction adipeuse : paramètres anthropométriques et indices de dysfonction métabolique. Les résultats montrent que dans le groupe de sujets étudiés (n=29 femmes, 15 témoins saines et 14 femmes avec résistance à l’insuline car atteintes du syndrome des ovaires polykystiques), certains indices de sensibilité à l’insuline pour la lipolyse dérivés de l’HGPO corrèlent bien avec ceux dérivés du clamp euglycémique hyperinsulinémique. Parmi ces indices, celui qui corrèle le mieux avec les indices du clamp et les paramètres anthropométriques et de dysfonction adipeuse est le T50[indice inférieur AGNE] (temps nécessaire pour diminuer de 50% le taux de base – à jeun – des AGNE). Nos résultats suggèrent donc que l’HGPO, facile à réaliser, peut être utilisée pour évaluer la sensibilité de la lipolyse à l’insuline. Nous pensons que la lipo-résistance à l’insuline peut être facilement quantifiée en clinique humaine.

Concentration dependence of sodium permeation and sodium ion interactions in the cyclic AMP-gated channels of mammalian olfactory receptor neurons

The dependence of currents through the cyclic nucleotide-gated (CNG) channels of mammalian olfactory receptor neurons (ORNs) on the concentration of NaCl was studied in excised inside-out patches from their dendritic knobs using the patch-clamp technique. With a saturating concentration (100 mu M) of adenosine 3', 5'-cyclic monophosphate (cAMP), the changes in the reversal potential of macroscopic currents were studied at NaCl concentrations from 25 to 300 mM. In symmetrical NaCl solutions without the addition of divalent cations, the current-voltage relations were almost linear, reversing close to O mV. When the external NaCl concentration was maintained at 150 mM and the internal concentrations were varied, the reversal potentials of the cAMP-activated currents closely followed the Na+ equilibrium potential indicating that P-Cl/P-Na approximate to 0. However, at low external NaCl concentrations (less than or equal to 100 mM) there was some significant chloride permeability. Our results further indicated that Na+ currents through these channels: (i) did not obey the independence principle; (ii) showed saturation kinetics with K(m)s in the range of 100-150 mM and (iii) displayed a lack of voltage dependence of conductance in asymmetric solutions that suggested that ion-binding sites were situated midway along the channel. Together, these characteristics indicate that the permeation properties of the olfactory CNG channels are significantly different from those of photoreceptor CNG channels.

Nitric oxide modulates the firing rate of the rat supraoptic magnocellular neurons

In vitro, nitric oxide (NO) inhibits the firing rate of magnocellular neurosecretory cells (MNCs) of hypothalamic supraoptic and paraventricular nuclei and this effect has been attributed to GABAergic activation. However, little is known about the direct effects of NO in MNCs. We used the patch-clamp technique to verify the effect Of L-arginine, a precursor for NO synthesis, and N-omega-nitro-L-arginine methyl ester hydrochloride (L-NAME), an inhibitor of NOS, on spontaneous electrical activity of MNCs after glutamatergic and GABAergic blockade in Wistar rat brain slices. 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 mu M) and DL-2-amino-5-phosphonovaleric acid (DL-AP5) (30 mu M) were used to block postsynaptic glutamatergic currents, and picrotoxin (30 mu M) and saclofen (30 mu M) to block ionotropic and metabotropic postsynaptic GABAergic currents. Under these conditions, 500 mu M L-arginine decreased the firing rate from 3.7 +/- 0.6 Hz to 1.3 +/- 0.3 Hz. Conversely, 100 mu M L-NAME increased the firing rate from 3.0 +/- 0.3 Hz to 5.8 +/- 0.4 Hz. All points histogram analysis showed changes in resting potential from -58.1 +/- 0.8 mV to -62.2 +/- 1.1 mV in the presence of L-arginine and from -59.8 +/- 0.7 mV to -56.9 +/- 0.8 mV by L-NAME. Despite the nitrergic modulator effect on firing rate, some MNCs had no significant changes in their resting potential. In those neurons, hyperpolarizing after-potential (HAP) amplitude increased from 12.4 +/- 1.2 mV to 16.8 +/- 0.7 mV by L-arginine, but without significant changes by L-NAME treatment. To our knowledge, this is the first demonstration that NO can inhibit MNCs independent of GABAergic inputs. Further, our results point to HAP as a potential site for nitrergic modulation. (C) 2008 IBRO. Published by Elsevier Ltd. All rights reserved.

Murine ventricular L-type Ca2+ current is enhanced by zinterol via beta(1)-adrenoceptors, and is reduced in TG4 mice overexpressing the human beta(2)-adrenoceptor

1 The functional coupling of B-2-adrenoceptors (beta (2)-ARs) to murine L-type Ca2+ current (I-Ca(L)) was investigated with two different approaches. The beta (2)-AR signalling cascade was activated either with the beta (2)-AR selective agonist zinterol (myocytes from wild-type mice), or by spontaneously active, unoccupied beta (2)-ARs (myocytes from TG4 mice with 435 fold overexpression of human beta (2)-ARs). Ca2+ and Ba2+ currents were recorded in the whole-cell and cell-attached configuration of the patch- clamp technique, respectively. 2 Zinterol (10 muM) significantly increased I-Ca(L) amplitude of wild-type myocytes by 19+/-5%, and this effect was markedly enhanced after inactivation of Gi-proteins with pertussis-toxin (PTX; 76+/-13% increase). However, the effect of zinterol was entirely mediated by the beta (1)-AR subtype, since it was blocked by the beta (1)-AR selective antagonist CGP 20712A (300 nM). The beta (2)-AR selective antagonist ICI 118,551 (50 nM) did not affect the response of I-Ca(L) to zinterol. 3 In myocytes with beta (2)-AR overexpression I-Ca(L) was not stimulated by the activated signalling cascade. On the contrary, I-Ca(L) was lower in TG4 myocytes and a significant reduction of single-channel activity was identified as a reason for the lower whole-cell I-Ca(L). The beta (2)-AR inverse agonist ICI 118,551 did not further decrease I-Ca(L). PTX-treatment increased current amplitude to values found in control myocytes. 4 In conclusion, there is no evidence for beta (2)-AR mediated increases of I-Ca(L) in wild-type mouse ventricular myocytes. Inactivation of Gi-proteins does not unmask beta (2)-AR responses to zinterol, but augments beta (1)-AR mediated increases of I-Ca(L). In the mouse model of beta (2)-AR overexpression I-Ca(L) is reduced due to tonic activation of Gi-proteins.

Ionic selectivity of native ATP-activated (P2X) receptor channels in dissociated neurones from rat parasympathetic ganglia

1. The relative permeability of the native P2X receptor channel to monovalent and divalent inorganic and organic cations was determined from reversal potential measurements of ATP-evoked currents in parasympathetic neurones dissociated from rat submandibular ganglia using the dialysed whole-cell patch clamp technique. 2. The P2X receptor-channel exhibited weak selectivity among the alkali metals with a selectivity sequence of Na+ > Li+ > Cs+ > Rb+ > K+, and permeability ratios relative to Cs+ (P-X/P-Cs) ranging from 1. 11 to 0.86. 3. The selectivity for the divalent alkaline earth cations was also weak with the sequence Ca2+ > Sr2+ > Ba2+ > Mn2+ > Mg2+. ATP-evoked currents were strongly inhibited when the extracellular divalent cation concentration was increased. 4, The calculated permeability ratios of different ammonium cations are higher than those of the alkali metal cations. The permeability sequence obtained for the saturated organic cations is inversely correlated with the size of the cation. The unsaturated organic cations have a higher permeability than that predicted by molecular size. 5. Acidification to pH 6.2 increased the ATP-induced current amplitude twofold, whereas alkalization to 8.2 and 9.2 markedly reduced current amplitude. Cell dialysis with either anti-P2X(2) and/or anti-P2X(4) but not anti-P2X(1) antibodies attenuated the ATP-evoked current amplitude. Taken together, these data are consistent with homomeric and/or heteromeric P2X(2) and P2X(4) receptor subtypes expressed in rat submandibular neurones. 6. The permeability ratios for the series of monovalent organic cations, with the exception of unsaturated cations, were approximately related to the ionic size. The relative permeabilities of the monovalent inoganic and organic cations tested are similar to those reported previously for cloned rat P2X2 receptors expressed in mammalian cells.

Basal nonselective cation permeability in rat cardiac microvascular endothelial cells

The presence of a basal nonselective cation permeability was mainly investigated in primary cultures of rat cardiac microvascular endothelial cells (CMEC) by applying both the patch-clamp technique and Fura-2 microfluorimetry. With low EGTA in the pipette solution, the resting membrane potential of CMEC was -21.2 +/- 1.1 mV, and a Ca2+-activated Cl- conductance was present. When the intracellular Ca2+ was buffered with high EGTA, the membrane potential decreased to 5.5 +/- 1.2 mV. In this condition, full or partial substitution of external Na+ by NMDG(+) proportionally reduced the inward component of the basal I-V relationship. This current was dependent on extracellular monovalent cations with a permeability sequence of K+ > Cs+ > Na+ > Li+ and was inhibited by Ca2+, La3+, Gd3+, and amiloride. The K+/Na+ permeability ratio, determined using the Goldman-Hodgkin-Katz equation, was 2.01. The outward component of the basal I-V relationship was reduced when intracellular K+ was replaced by NMDG(+), but was not sensitive to substitution by Cs+. Finally, microfluorimetric experiments indicated the existence of a basal Ca2+ entry pathway, inhibited by La3+ and Gd3+. The basal nonselective cation permeability in CMEC could be involved both in the control of myocardial ionic homeostasis, according to the model of the blood-heart barrier, and in the modulation of Ca2+ -dependent processes. (C) 2002 Elsevier Science (USA).

Electrical Properties of Isolated Cardiomyocytes in a Rat Model of Thiamine Deficiency

In modern society, thiamine deficiency (TD) remains an important medical condition linked to altered cardiac function. There have been contradictory reports about the impact of TD on heart physiology, especially in the context of cardiac excitability. In order to address this particular question, we used a TD rat model and patch-clamp technique to investigate the electrical properties of isolated cardiomyocytes from epicardium and endocardium. Neither cell type showed substantial differences on the action potential waveform and transient outward potassium current. Based on our results we can conclude that TD does not induce major electrical remodeling in isolated cardiac myocytes in either endocardium or epicardium cells.

Effect of somatostatin on duodenal glucose absorption in man.

OBJECTIVE: The hyperglycemic hyperinsulinemic clamp technique using intraduodenally infused glucose is an attractive tool for studying postprandial glucose metabolism under strictly controlled conditions. Because it requires the use of somatostatin (SST), we examined, in this study, the effect of SST on intestinal glucose absorption. CONTEXT: Twenty-six normal volunteers were given a constant 3-h intraduodenal infusion of glucose (6 mg.kg(-1).min(-1)) labeled with [2-(3)H]glucose for glucose absorption measurement. During glucose infusion, 19 subjects received iv SST at doses of 10-100 ng.kg(-1).min(-1) plus insulin and glucagon, and seven subjects were studied under control conditions. In the controls, glucose was absorbed at a rate that, after a 20-min lag period, equaled the infusion rate. RESULTS: With all the doses of SST tested, absorption was considerably delayed but equaled the rate of infusion after 3 h. At that time, only 5 +/- 2% of the total amount of infused glucose was unabsorbed in the control subjects vs. 36 +/- 2% (P < 0.001) in the SST-infused subjects. In the latter, the intraluminal residue was almost totally absorbed within 40 min of the cessation of SST infusion. At the lowest dose of SST tested (10 ng.kg(-1).min(-1)), suppression of insulin secretion was incomplete. CONCLUSION: These properties of SST hamper the use of intraduodenal hyperglycemic hyperinsulinemic clamps as a tool for exploring postprandial glucose metabolism.