Patch-clamp detection of macromolecular translocation along nuclear pores

The present paper reviews the application of patch-clamp principles to the detection and measurement of macromolecular translocation along the nuclear pores. We demonstrate that the tight-seal 'gigaseal' between the pipette tip and the nuclear membrane is possible in the presence of fully operational nuclear pores. We show that the ability to form a gigaseal in nucleus-attached configurations does not mean that only the activity of channels from the outer membrane of the nuclear envelope can be detected. Instead, we show that, in the presence of fully operational nuclear pores, it is likely that the large-conductance ion channel activity recorded derives from the nuclear pores. We conclude the technical section with the suggestion that the best way to demonstrate that the nuclear pores are responsible for ion channel activity is by showing with fluorescence microscopy the nuclear translocation of ions and small molecules and the exclusion of the same from the cisterna enclosed by the two membranes of the envelope. Since transcription factors and mRNAs, two major groups of nuclear macromolecules, use nuclear pores to enter and exit the nucleus and play essential roles in the control of gene activity and expression, this review should be useful to cell and molecular biologists interested in understanding how patch-clamp can be used to quantitate the translocation of such macromolecules into and out of the nucleus

Inibição da corrente de cálcio tipo L por tramadol e enantiômeros em miócitos cardíacos de ratos

FUNDAMENTO: O tramadol é um analgésico de ação central cujo mecanismo de ação envolve a ativação de um receptor opioide. Anteriormente, mostramos que o tramadol e seus enantiômeros apresentavam um efeito inotrópico negativo sobre o músculo papilar no qual o (+)-enantiômero era mais potente que (-)- e (±)-tramadol. OBJETIVO: No presente trabalho, investigamos os efeitos do tramadol e seus enantiômeros na corrente de cálcio tipo L (I Ca-L). MÉTODOS: Os experimentos foram realizados em miócitos ventriculares isolados de ratos Wistar utilizando a técnica de patch-clamp com configuração de célula inteira. RESULTADOS: O tramadol (200 µM) reduziu a amplitude de pico do I Ca-L em potenciais de 0 a +50 mV. Em 0 mV, a I Ca-L foi reduzida em 33,7 ± 7,2%. (+)- e (-)-tramadol (200 µM) produziram uma inibição semelhante da I Ca-L, na qual a amplitude do pico foi reduzida em 64,4 ± 2,8% e 68,9 ± 5,8%, respectivamente a 0 mV (P > 0,05). O tramadol, (+)- e (-)-tramadol mudaram a inativação de estado estacionário de I Ca-L para potenciais de membrana mais negativos. Além disso, tramadol e (+)-tramadol alteraram significativamente a curva de recuperação dependente de tempo da I Ca-L para a direita e reduziram a recuperação de I Ca-L da inativação. A constante de tempo foi aumentada de 175,6 ± 18,6 a 305,0 ± 32,9 ms (P < 0,01) para o tramadol e de 248,1 ± 28,1 ms para 359,0 ± 23,8 ms (P < 0,05) para o (+)-tramadol. O agonista do receptor µ-opioide (DAMGO) não tem nenhum efeito na I Ca-L. CONCLUSÃO: A inibição da I Ca-L induzida por tramadol e seus enantiômeros não teve relação com a ativação de receptores opioides e poderia explicar, pelo menos em parte, seu efeito inotrópico negativo cardíaco.

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.

Cardiomyocyte dysfunction during the chronic phase of Chagas disease

Chagas disease, which is caused by the parasite Trypanosoma cruzi, is an important cause of heart failure. We investigated modifications in the cellular electrophysiological and calcium-handling characteristics of an infected mouse heart during the chronic phase of the disease. The patch-clamp technique was used to record action potentials (APs) and L-type Ca2+ and transient outward K+ currents. [Ca2+]i changes were determined using confocal microscopy. Infected ventricular cells showed prolonged APs, reduced transient outward K+ and L-type Ca2+ currents and reduced Ca2+ release from the sarcoplasmic reticulum. Thus, the chronic phase of Chagas disease is characterised by cardiomyocyte dysfunction, which could lead to heart failure.

Connexin hemichannels and cell-cell channels: comparison of properties

Connexin46 (Cx46) forms functional hemichannels in the absence of contact by an apposed hemichannel and we have used these hemichannels to study gating and permeation at the single channel level with high time resolution. Using both cell-attached and -excised patch configurations, we find that single Cx46 hemichannels exhibit some properties expected of half of a gap junction channel, as well as novel properties. Cx46 hemichannels have a large unitary conductance (~300 pS) and a relatively large pore as inferred from permeability to TEA. Both monovalent cations and anions can permeate, but cations are substantially more permeable. The open channel conductance shows marked inward rectification in symmetric salts. We find that the conductance and permeability properties of Cx46 cell-cell channels can be explained by the series addition of two hemichannels. These data suggest that the pore structures of unapposed hemichannels and cell-cell channels are conserved. Also like cell-cell channels, unapposed Cx46 hemichannels are closed by elevated levels of H+ or Ca2+ ions on the cytoplasmic face. Closure occurs in excised patches indicating that the actions of these agents do not require a soluble cytoplasmic factor. Fast (<0.5 ms) application of H+ to either side of the open hemichannel causes an immediate small reduction in unitary conductance followed by complete closure with latencies that are dependent on H+ concentration and side of application; sensitivity is much greater to H+ on the cytoplasmic side. Closure by cytoplasmic H+ does not require that the hemichannel be open. Thus, H+ ions readily permeate Cx46 hemichannels, but at high enough concentration close them by acting at a cytoplasmic site(s) that causes a conformational change resulting in complete closure. Extracellular H+ may permeate to act on the cytoplasmic site or act on a lower affinity extracellular site. Thus, the unapposed hemichannel is a valuable tool in addressing fundamental questions concerning the operation of gap junction channels that are difficult to answer by existing methods. The ability of Cx46, and perhaps other connexins, to form functional unapposed hemichannels that are opened by moderate depolarization may represent an unexplored role of connexins as mediators of transport across the plasma membrane.

Modulation of intercellular communication by differential regulation and heteromeric mixing of co-expressed connexins

Intercellular communication may be regulated by the differential expression of subunit gap junction proteins (connexins) which form channels with differing gating and permeability properties. Endothelial cells express three different connexins (connexin37, connexin40, and connexin43) in vivo. To study the differential regulation of expression and synthesis of connexin37 and connexin43, we used cultured bovine aortic endothelial cells which contain these two connexins in vitro. RNA blots demonstrated discordant expression of these two connexins during growth to confluency. RNA blots and immunoblots showed that levels of these connexins were modulated by treatment of cultures with transforming growth factor-ß1. To examine the potential ability of these connexins to form heteromeric channels (containing different connexins within the same hemi-channel), we stably transfected connexin43-containing normal rat kidney (NRK) cells with connexin37 or connexin40. In the transfected cells, both connexin proteins were abundantly produced and localized in identical distributions as detected by immunofluorescence. Double whole-cell patch-clamp studies showed that co-expressing cells exhibited unitary channel conductances and gating characteristics that could not be explained by hemi-channels formed of either connexin alone. These observations suggest that these connexins can readily mix with connexin43 to form heteromeric channels and that the intercellular communication between cells is determined not only by the properties of individual connexins, but also by the interactions of those connexins to form heteromeric channels with novel properties. Furthermore, modulation of levels of the co-expressed connexins during cell proliferation or by cytokines may alter the relative abundance of different heteromeric combinations.

Impaired beta-adrenergic response and decreased L-type calcium current of hypertrophied left ventricular myocytes in postinfarction heart failure

Infarct-induced heart failure is usually associated with cardiac hypertrophy and decreased ß-adrenergic responsiveness. However, conflicting results have been reported concerning the density of L-type calcium current (I Ca(L)), and the mechanisms underlying the decreased ß-adrenergic inotropic response. We determined I Ca(L) density, cytoplasmic calcium ([Ca2+]i) transients, and the effects of ß-adrenergic stimulation (isoproterenol) in a model of postinfarction heart failure in rats. Left ventricular myocytes were obtained by enzymatic digestion 8-10 weeks after infarction. Electrophysiological recordings were obtained using the patch-clamp technique. [Ca2+]i transients were investigated via fura-2 fluorescence. ß-Adrenergic receptor density was determined by [³H]-dihydroalprenolol binding to left ventricle homogenates. Postinfarction myocytes showed a significant 25% reduction in mean I Ca(L) density (5.7 ± 0.28 vs 7.6 ± 0.32 pA/pF) and a 19% reduction in mean peak [Ca2+]i transients (0.13 ± 0.007 vs 0.16 ± 0.009) compared to sham myocytes. The isoproterenol-stimulated increase in I Ca(L) was significantly smaller in postinfarction myocytes (Emax: 63.6 ± 4.3 vs 123.3 ± 0.9% in sham myocytes), but EC50 was not altered. The isoproterenol-stimulated peak amplitude of [Ca2+]i transients was also blunted in postinfarction myocytes. Adenylate cyclase activation through forskolin produced similar I Ca(L) increases in both groups. ß-Adrenergic receptor density was significantly reduced in homogenates from infarcted hearts (Bmax: 93.89 ± 20.22 vs 271.5 ± 31.43 fmol/mg protein in sham myocytes), while Kd values were similar. We conclude that postinfarction myocytes from large infarcts display reduced I Ca(L) density and peak [Ca2+]i transients. The response to ß-adrenergic stimulation was also reduced and was probably related to ß-adrenergic receptor down-regulation and not to changes in adenylate cyclase activity.

Electrophysiological evidence for glial-subtype glutamate transporter functional expression in rat cerebellar granule neurons

A glutamate-sensitive inward current (Iglu) is described in rat cerebellar granule neurons and related to a glutamate transport mechanism. We examined the features of Iglu using the patch-clamp technique. In steady-state conditions the Iglu measured 8.14 ± 1.9 pA. Iglu was identified as a voltage-dependent inward current showing a strong rectification at positive potentials. L-Glutamate activated the inward current in a dose-dependent manner, with a half-maximal effect at about 18 µM and a maximum increase of 51.2 ± 4.4%. The inward current was blocked by the presence of dihydrokainate (0.5 mM), shown by others to readily block the GLT1 isoform. We thus speculate that Iglu could be attributed to the presence of a native glutamate transporter in cerebellar granule neurons.

Differentiation of autonomic reflex control begins with cellular mechanisms at the first synapse within the nucleus tractus solitarius

Visceral afferents send information via cranial nerves to the nucleus tractus solitarius (NTS). The NTS is the initial step of information processing that culminates in homeostatic reflex responses. Recent evidence suggests that strong afferent synaptic responses in the NTS are most often modulated by depression and this forms a basic principle of central integration of these autonomic pathways. The visceral afferent synapse is uncommonly powerful at the NTS with large unitary response amplitudes and depression rather than facilitation at moderate to high frequencies of activation. Substantial signal depression occurs through multiple mechanisms at this very first brainstem synapse onto second order NTS neurons. This review highlights new approaches to the study of these basic processes featuring patch clamp recordings in NTS brain slices and optical techniques with fluorescent tracers. The vanilloid receptor agonist, capsaicin, distinguishes two classes of second order neurons (capsaicin sensitive or capsaicin resistant) that appear to reflect unmyelinated and myelinated afferent pathways. The differences in cellular properties of these two classes of NTS neurons indicate clear functional differentiation at both the pre- and postsynaptic portions of these first synapses. By virtue of their position at the earliest stage of these pathways, such mechanistic differences probably impart important differentiation in the performance over the entire reflex pathways.

Electrophysiological evidence for the presence of NR2C subunits of N-methyl-D-aspartate receptors in rat neurons of the nucleus tractus solitarius

The nucleus tractus solitarius (NTS) plays an important role in the control of autonomic reflex functions. Glutamate, acting on N-methyl-D-aspartate (NMDA) and non-NMDA ionotropic receptors, is the major neurotransmitter in this nucleus, and the relative contribution of each receptor to signal transmission is unclear. We have examined NMDA excitatory postsynaptic currents (NMDA-EPSCs) in the subpostremal NTS using the whole cell patch clamp technique on a transverse brainstem slice preparation. The NMDA-EPSCs were evoked by stimulation of the solitary tract over a range of membrane potentials. The NMDA-EPSCs, isolated pharmacologically, presented the characteristic outward rectification and were completely blocked by 50 µM DL-2-amino-5-phosphonopentanoic acid. The I-V relationship of the NMDA response shows that current, with a mean (± SEM) amplitude of -41.2 ± 5.5 pA, is present even at a holding potential of -60 mV, suggesting that the NMDA receptors are weakly blocked by extracellular Mg2+ at near resting membrane potentials. This weak block can also be inferred from the value of 0.67 ± 0.17 for parameter delta obtained from a fit of the Woodhull equation to the I-V relationship. The maximal inward current measured on the I-V relationship was at -38.7 ± 4.2 mV. The decay phase of the NMDA currents was fitted with one exponential function with a decay time constant of 239 ± 51 and 418 ± 80 ms at a holding potential of -60 and +50 mV, respectively, which became slower with depolarization (e-fold per 145 mV). The biophysical properties of the NMDA receptors observed in the present study suggest that these receptors in the NTS contain NR2C subunits and may contribute to the synaptic signal integration.

Relationships between dendritic morphology, spatial distribution and firing patterns in rat layer 1 neurons

The cortical layer 1 contains mainly small interneurons, which have traditionally been classified according to their axonal morphology. The dendritic morphology of these cells, however, has received little attention and remains ill defined. Very little is known about how the dendritic morphology and spatial distribution of these cells may relate to functional neuronal properties. We used biocytin labeling and whole cell patch clamp recordings, associated with digital reconstruction and quantitative morphological analysis, to assess correlations between dendritic morphology, spatial distribution and membrane properties of rat layer 1 neurons. A total of 106 cells were recorded, labeled and subjected to morphological analysis. Based on the quantitative patterns of their dendritic arbor, cells were divided into four major morphotypes: horizontal, radial, ascendant, and descendant cells. Descendant cells exhibited a highly distinct spatial distribution in relation to other morphotypes, suggesting that they may have a distinct function in these cortical circuits. A significant difference was also found in the distribution of firing patterns between each morphotype and between the neuronal populations of each sublayer. Passive membrane properties were, however, statistically homogeneous among all subgroups. We speculate that the differences observed in active membrane properties might be related to differences in the synaptic input of specific types of afferent fibers and to differences in the computational roles of each morphotype in layer 1 circuits. Our findings provide new insights into dendritic morphology and neuronal spatial distribution in layer 1 circuits, indicating that variations in these properties may be correlated with distinct physiological functions.

Estragole blocks neuronal excitability by direct inhibition of Na+ channels

Estragole is a volatile terpenoid, which occurs naturally as a constituent of the essential oils of many plants. It has several pharmacological and biological activities. The objective of the present study was to investigate the mechanism of action of estragole on neuronal excitability. Intact and dissociated dorsal root ganglion neurons of rats were used to record action potential and Na+ currents with intracellular and patch-clamp techniques, respectively. Estragole blocked the generation of action potentials in cells with or without inflexions on their descendant (repolarization) phase (Ninf and N0 neurons, respectively) in a concentration-dependent manner. The resting potentials and input resistances of Ninf and N0 cells were not altered by estragole (2, 4, and 6 mM). Estragole also inhibited total Na+ current and tetrodotoxin-resistant Na+ current in a concentration-dependent manner (IC50 of 3.2 and 3.6 mM, respectively). Kinetic analysis of Na+ current in the presence of 4 mM estragole showed a statistically significant reduction of fast and slow inactivation time constants, indicating an acceleration of the inactivation process. These data demonstrate that estragole blocks neuronal excitability by direct inhibition of Na+ channel conductance activation. This action of estragole is likely to be relevant to the understanding of the mechanisms of several pharmacological effects of this substance.

Preserved xenogenic amniotic membrane as a patch on the repair of superficial corneal ulcers in rabbits

The aim of this study was to evaluate the effects of canine amniotic membrane, previously preserved in glycerin, used as a patch on the repair of experimentally-made superficial corneal ulcers and to compare corneal epithelization between the treated and non-treated groups. Xenogeneic amniotic membranes were collected aseptically and preserved in 99% glycerin at room temperature. Each animal was anesthetized and submitted to superficial corneal keratectomy of the left eye. The treated group received a fragment of canine amniotic membrane as a patch, while the control group had no treatment. The treated group showed blepharospasm, ocular discharge and conjunctival congestion. The membrane accelerated corneal repair in the beginning of the process, however, it delayed its conclusion (p<0.05). Treated eyes showed greater vessel formation and decreased corneal transparency (p<0.05). The stroma of the control group was thicker than that of the treated group (p<0.05). We suggest that amniotic membrane used in this manner can be applied as a therapy for superficial corneal ulcers in the beginning phases of the repair process.

Ecological aspects of American cutaneous leishmaniasis: 6. Man-biting sandfly fauna of a patch of residual forest in the middle and north-eastern region of the São Paulo State, Brazil

This report shows the phlebotomine vectorial role in the endemic cutaneous leishmaniasis situated in the middle and north-eastern region of the São Paulo, Brazil. Analysis of information encloses a sandfly man-biting fauna of four different patches of residual forest. So, using the human-bait and Shannon trap for a period of one year we have caught 16,869 sandflies. The predominance of Lutzomyia intermedia (85,6%) was clear. Moreover, the low density of Lutzomyia whitmani and Lutzomyia pessoai and their epidemiological implications at present and in the past are discussed. Information about the daily activity of some species is given and we emphasize for the first time, the diurnal activity of Lutzomyia firmatoi in the São Paulo State.

Late Results of Endoventricular Patch Plasty Repair in Akinetic and Dyskinetic Areas After Acute Myocardial Infarction

OBJECTIVE - To assess the surgical results of endoventricular patch plasty repair in akinetic and dyskinetic left ventricular areas. METHODS - We studied 52 patients who had undergone endoventricular patch plasty repair associated with myocardial revascularization. The preoperative functional class distribution was as follows: class I in 1 (1.9%) patient; class II in 2 (3.8%) patients; class III in 23 (44.2%) patients; and class IV in 26 (50%) patients. RESULTS - The immediate mortality rate was 7.6% (4 patients). The clinical outcome of 44 patients followed up within a mean postoperative time of 29±25 months was as follows: class I in 33 (75%) patients; class II in 7 (15.9%) patients; class III in 2 (4.5%) patients; and class IV in 2 (4.5%) patients. Comparison between pre- and postoperative catheterization in 21 patients showed that the ejection fraction increased from 46.3% to 51.3% (p=0. 17); the left ventricular systolic volume decreased from 76.4 mL to 57.5 mL, (p=0.078); and the left ventricular diastolic volume decreased from 141.2 mL to 105.8 mL (p=0.0 73). These findings showed the tendency toward improvement, but with nonsignificant results. CONCLUSION - The technique proved to be effective, to have a low mortality rate, to cause significant clinical improvement, an increase in ejection fraction, and a reduction in left ventricular volumes.