Karyotype evolution in the genus Jacaranda Juss. (Jacarandeae, Bignoniaceae): chromosome numbers and heterochromatin.

Most taxa in the Bignoniaceae have 2n = 40, but the basal clade Jacarandeae has 2n = 36, suggesting that x = 18 is the ancestral basic number for the family. Variations in heterochromatin band patterns in genera that are numerically stable, such as Jacaranda, could facilitate our understanding of the chromosomal and karyotypic evolution of the family. We characterized heterochromatin distributions in six Jacaranda species using chromomycin A3 (CMA) and 4'6-diamidino-2-phenylindole (DAPI). All of them had 2n = 36, including first counts for Jacaranda bracteata Bureau & K. Schum., Jacaranda irwinii A.H. Gentry, Jacaranda jasminoides (Thunb.) Sandwith, and Jacaranda rugosa A.H. Gentry. Their karyotypes had four to eight terminal CMA+/DAPI- bands per monoploid set. In the section Monolobos, Jacaranda brasiliana (Lam.) Pers. had eight terminal bands and Jacaranda mimosifolia D. Don had four; in the section Dilobos, J. bracteata had six bands per monoploid set, with the other species having five. While three species in the section Dilobos had the same number of terminal bands, J. irwinii had two additional pericentromeric bands and a proximal heterozygotic band, and J. bracteata had two distended CMA bands. The consistent records of 2n = 36 in Jacaranda may represent a plesiomorphic condition for the Bignoniaceae; therefore, the family originated from an ancestor with x = 18. However, 2n = 36 may represent a derived condition, and the family could have had an ancestral basic number of x = 20 that is still conserved in most representatives of the family.

Rehabilitation of atrophic anophthalmic cavity with orthostatic ocular prosthesis: A clinical report.

The absence of the eyeball can generate psychosocial and facial harmony changes, such as atrophy of the muscles around it. In these cases, the use of an orthostatic prosthesis with expanding function fosters distension of the tissues for subsequent rehabilitation. This technique consists of making individual ocular prostheses with gradual enlargement of size. The aim of this following clinical report was to describe the technique used in the standing prosthetic rehabilitation of a patient, 73 years old, who underwent enucleation of the right eye as a result of glaucoma. Clinical and laboratory procedures were performed such as impression, adjusting curvature of the sclera, centering the pupil area and processing in heat-cured acrylic resin three prostheses made according to the expansion of the anophthalmic cavity. At the end of treatment, there was a considerable increase of the cavity, allowing for volume replacement similar to that existing in the patient's contralateral orbit, thus generating a satisfactory facial harmony.

Bladder pain induced by prolonged peripheral alpha 1A adrenoceptor stimulation involves the enhancement of transient receptor potential vanilloid 1 activity and an increase of urothelial adenosine triphosphate release.

AIM: Pathophysiological mechanisms of chronic visceral pain (CVP) are unknown. This study explores the association between the sympathetic system and bladder nociceptors activity by testing the effect of a prolonged adrenergic stimulation on transient receptor potential vanilloid 1 (TRPV1) activity and on urothelial adenosine triphosphate (ATP) release. METHODS: Female Wistar rats received saline, phenylephrine (PHE), PHE + silodosin, PHE + naftopidil or PHE + prazosin. TRPV1 knockout and wild-type mice received saline or PHE. Visceral pain behaviour tests were performed before and after treatment. Cystometry was performed, during saline and capsaicin infusion. Fos immunoreactivity was assessed in L6 spinal cord segment. Human urothelial ATP release induced by mechanical and thermal stimulation was evaluated. RESULTS: Subcutaneous, but not intrathecal, PHE administration induced pain, which was reversed by silodosin, a selective alpha 1A adrenoceptor antagonist, but not by naftopidil, a relatively selective antagonist for alpha 1D adrenoceptor. Silodosin also reversed PHE-induced bladder hyperactivity and L6 spinal cord Fos expression. Thus, in subsequent experiments, only silodosin was used. Wild-type, but not TRPV1 knockout, mice exhibited phenylephrine-induced pain. Capsaicin induced a greater increase in voiding contractions in PHE-treated rats than in control animals, and silodosin reversed this effect. When treated with PHE, ATP release from human urothelial cells was enhanced either by mechanical stimulation or by lowering the thermal threshold of urothelial TRPV1, which becomes abnormally responsive at body temperature. CONCLUSION: This study suggests that the activation of peripheral alpha 1A adrenoceptors induces CVP, probably through its interaction with TRPV1 and ATP release.

P2X7 receptor activation downmodulates Na(+)-dependent high-affinity GABA and glutamate transport into rat brain cortex synaptosomes.

Sodium-dependent high-affinity amino-acid transporters play crucial roles in terminating synaptic transmission in the central nervous system (CNS). However, there is lack of information about the mechanisms underlying the regulation of amino-acid transport by fast-acting neuromodulators, like ATP. Here, we investigated whether activation of the ATP-sensitive P2X7 receptor modulates Na(+)-dependent high-affinity γ-aminobutyric acid (GABA) and glutamate uptake into nerve terminals (synaptosomes) of the rat cerebral cortex. Radiolabeled neurotransmitter accumulation was evaluated by liquid scintillation spectrometry. The cell-permeant sodium-selective fluorescent indicator, SBFI-AM, was used to estimate Na(+) influx across plasma membrane. 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP, 3-300 µM), a prototypic P2X7 receptor agonist, concentration-dependently decreased [(3)H]GABA (14%) and [(14)C]glutamate (24%) uptake; BzATP decreased transport maximum velocity (Vmax) without affecting the Michaelis constant (Km) values. The selective P2X7 receptor antagonist, A-438079 (3 µM), prevented inhibition of [(3)H]GABA and [(14)C]glutamate uptake by BzATP (100 µM). The inhibitory effect of BzATP coincided with its ability to increase intracellular Na(+) and was mimicked by Na(+) ionophores, like gramicidin and monensin. Increases in intracellular Na(+) (with veratridine or ouabain) or substitution of extracellular Na(+) by N-methyl-D-glucamine (NMDG)(+) all decreased [(3)H]GABA and [(14)C]glutamate uptake and attenuated BzATP effects. Uptake inhibition by BzATP (100 µM) was also attenuated by calmidazolium, which selectively inhibits Na(+) currents through the P2X7 receptor pore. In conclusion, disruption of the Na(+) gradient by P2X7 receptor activation downmodulates high-affinity GABA and glutamate uptake into rat cortical synaptosomes. Interference with amino-acid transport efficacy may constitute a novel target for therapeutic management of cortical excitability.

Extracellular calcium triggers unique transcriptional programs and modulates staurosporine-induced cell death in Neurospora crassa.

Alterations in the intracellular levels of calcium are a common response to cell death stimuli in animals and fungi and, particularly, in the Neurospora crassa response to staurosporine. We highlight the importance of the extracellular availability of Ca2+ for this response. Limitation of the ion in the culture medium further sensitizes cells to the drug and results in increased accumulation of reactive oxygen species (ROS). Conversely, an approximately 30-fold excess of external Ca2+ leads to increased drug tolerance and lower ROS generation. In line with this, distinct staurosporine-induced cytosolic Ca2+ signaling profiles were observed in the absence or presence of excessive external Ca2+. High-throughput RNA sequencing revealed that different concentrations of extracellular Ca2+ define distinct transcriptional programs. Our transcriptional profiling also pointed to two putative novel Ca2+-binding proteins, encoded by the NCU08524 and NCU06607 genes, and provides a reference dataset for future investigations on the role of Ca2+ in fungal biology.

Conhecimento botânico medicinal sobre espécies vegetais nativas da caatinga e plantas espontâneas no agreste da Paraíba, Brasil / Botanical medical knowledge of native species of the Caatinga and spontaneous plants in the Agreste region of the state of Paraíba, Brazil

O bioma Caatinga apresenta diversas espécies vegetais amplamente empregadas pelas populações rurais, especialmente na fitoterapia, abrangendo diversos usos no tratamento de determinadas enfermidades. As plantas espontâneas, apesar de serem entendidas como espécies daninhas ou invasoras, concomitantemente apresentam propriedades fitoquímicas que podem ser aproveitadas no âmbito medicinal. Nesta concepção, o referente trabalho tem como objetivo identificar espécies vegetais nativas da Caatinga, assim como plantas espontâneas, empregadas na medicina popular através de estudo etnobotânico desenvolvido na zona rural do município de Serra da Raiz, Agreste da Paraíba, Nordeste do Brasil. O levantamento das plantas de uso fitoterápico foi estabelecido através de questionamentos e entrevistas semiestruturadas com 57 famílias da região. Foram coletadas informações referentes a 55 espécies vegetais e seus empregos terapêuticos, destacando-se entre elas: Myracrodruom urundeuva Allemão (Aroeira), Genipa americana L. (Jenipapo), Solanum paniculatum L. (Jurubeba) e Anadenanthera colubrina (Vell.) Brenan (Angico) por serem amplamente utilizadas no tratamento de diversas enfermidades pelos moradores locais.

The Brazilian Caatinga has several plant species widely used by rural populations, especially in phytotherapy, covering many uses in the treatment of diseases. The spontaneous plants, although regarded as invasive plants or weeds, present phytochemical properties that can be exploited in medicine. This study aims to identify native plant species of the Caatinga and spontaneous plants used in medicine through an ethnobotanical study developed in the municipality of Serra da Raiz, Agreste area of the state of Paraíba, Brazil. The survey of plants used in herbal medicine was established through the questioning of and semi-structured interviews with 57 families in the region. Information was record on 55 plant species and their therapeutic uses. The species most used in the treatment of various diseases were Myracrodruon urundeuva Allemão, Genipa americana L., Solanum paniculatum L. and Anadenanthera colubrina (Vell.) Brenan..

Effect of the I(to) activator NS5806 on cloned K(V)4 channels depends on the accessory protein KChIP2.

BACKGROUND AND PURPOSE: The compound NS5806 increases the transient outward current (I(to)) in canine ventricular cardiomyocytes and slows current decay. In human and canine ventricle, I(to) is thought to be mediated by K(V)4.3 and various ancillary proteins, yet, the exact subunit composition of I(to) channels is still debated. Here we characterize the effect of NS5806 on heterologously expressed putative I(to) channel subunits and other potassium channels. EXPERIMENTAL APPROACH: Cloned K(V)4 channels were co-expressed with KChIP2, DPP6, DPP10, KCNE2, KCNE3 and K(V)1.4 in Xenopus laevis oocytes or CHO-K1 cells. KEY RESULTS: NS5806 increased K(V)4.3/KChIP2 peak current amplitudes with an EC(50) of 5.3 +/- 1.5microM and significantly slowed current decay. KCNE2, KCNE3, DPP6 and DPP10 modulated K(V)4.3 currents and the response to NS5806, but current decay was slowed only in complexes containing KChIP2. The effect of NS5806 on K(V)4.2 was similar to that on K(V)4.3, and current decay was only slowed in presence of KChIP2. However, for K(V)4.1, the slowing of current decay by NS5806 was independent of KChIP2. K(V)1.4 was strongly inhibited by 10 microM NS5806 and K(V)1.5 was inhibited to a smaller extent. Effects of NS5806 on kinetics of currents generated by K(V)4.3/KChIP2/DPP6 with K(V)1.4 in oocytes could reproduce those on cardiac I(to) in canine ventricular myocytes. K(V)7.1, K(V)11.1 and K(ir)2 currents were unaffected by NS5806. CONCLUSION AND IMPLICATIONS: NS5806 modulated K(V)4 channel gating depending on the presence of KChIP2, suggesting that NS5806 can potentially be used to address the molecular composition as well as the physiological role of cardiac I(to).

hERG1a/1b heteromeric currents exhibit amplified attenuation of inactivation in variant 1 short QT syndrome.

Potassium channels encoded by hERG (human ether-à-go-go-related gene) underlie the cardiac rapid delayed rectifier K+ current (IKr) and hERG mutations underpin clinically important repolarization disorders. Virtually all electrophysiological investigations of hERG mutations have studied exclusively the hERG1a isoform; however, recent evidence indicates that native IKr channels may be comprised of hERG1a together with the hERG1b variant, which has a shorter N-terminus. Here, for the first time, electrophysiological effects were studied of a gain-of-function hERG mutation (N588K; responsible for the 'SQT1' variant of the short QT syndrome) on current (I(hERG1a/1b)) carried by co-expressed hERG1a/1b channels. There were no significant effects of N588K on I(hERG1a/1b) activation or deactivation, but N588K I(hERG1a/1b) showed little inactivation up to highly positive voltages (< or = +80 mV), a more marked effect than seen for hERG1a expressed alone. I(hERG1a/1b) under action potential voltage-clamp, and the effects on this of the N588K mutation, also showed differences from those previously reported for hERG1a. The amplified attenuation of I(hERG) inactivation for the N588K mutation reported here indicates that the study of co-expressed hERG1a/1b channels should be considered when investigating clinically relevant hERG channel mutations, even if these reside outside of the N-terminus region.

Comparative effects of the short QT N588K mutation at 37 degrees C on hERG K+ channel current during ventricular, Purkinje fibre and atrial action potentials: an action potential clamp study.

The short QT syndrome (SQTS) is a cardiac repolarisation disorder characterised by abbreviated QT intervals on the electrocardiogram and by an increased risk of atrial and ventricular arrhythmias and sudden death. The SQT1 variant involves a gain-of-function mutation (N588K) that impairs inactivation of the hERG (human ether-a-go-go-related gene) potassium channel and, thereby, increases current mediated by the rapid delayed rectifier potassium current (I(Kr)) in the heart. Here, the action potential voltage clamp (AP clamp) technique was applied to Chinese Hamster Ovary cells expressing wild-type or N588K-hERG at 37 degrees C, to compare effects of the N588K mutation on hERG current (I(hERG)) during ventricular, atrial and Purkinje fibre APs. The N588K mutation altered the I(hERG) profile during each AP type; increased maximal repolarising current occurred earlier during AP repolarisation (with shifts of +60 mV, +30 mV and +15 mV respectively for ventricular, Purkinje fibre and atrial APs). Thus SQT1 may influence repolarising I(hERG) for each cell type, with AP clamp experiments and simulation data indicating the greatest effect during ventricular APs. Changes in the timing of outward I(hERG) transients elicited by premature stimuli following AP commands indicate that SQT1 may alter the protection that hERG provides cardiac tissue against premature arrhythmogenic stimuli.

Functionally distinct sodium channels in ventricular epicardial and endocardial cells contribute to a greater sensitivity of the epicardium to electrical depression.

A greater depression of the action potential (AP) of the ventricular epicardium (Epi) versus endocardium (Endo) is readily observed in experimental models of acute ischemia and Brugada syndrome. Endo and Epi differences in transient outward K(+) current and/or ATP-sensitive K(+) channel current are believed to contribute to the differential response. The present study tested the hypothesis that the greater sensitivity of Epi is due in part to its functionally distinct early fast Na(+) current (I(Na)). APs were recorded from isolated Epi and Endo tissue slices and coronary-perfused wedge preparations before and after exposures to elevated extracellular K(+) concentration ([K(+)](o); 6-12 mM). I(Na) was recorded from Epi and Endo myocytes using whole cell patch-clamp techniques. In tissue slices, increasing [K(+)](o) to 12 mM reduced V(max) to 51.1 +/- 5.3% and 26.8 +/- 9.6% of control in Endo (n = 9) and Epi (n = 14), respectively (P < 0.05). In wedge preparations (n = 12), the increase in [K(+)](o) caused selective depression of Epi APs and transmural conduction slowing and block. I(Na) density was not significantly different between Epi (n = 14) and Endo (n = 15) cells, but Epi cells displayed a more negative half-inactivation voltage [-83.6 +/- 0.1 and -75.5 +/- 0.3 mV for Epi (n = 16) and Endo (n = 16), respectively, P < 0.05]. Our data suggest that reduced I(Na) availability in ventricular Epi may contribute to its greater sensitivity to electrical depression and thus may contribute to the R-ST segment changes observed under a variety of clinical conditions including acute myocardial ischemia, severe hyperkalemia, and Brugada syndrome.

Modulation of I(Kr) inactivation by mutation N588K in KCNH2: a link to arrhythmogenesis in short QT syndrome.

OBJECTIVE: Short QT syndrome (SQTS) is characterized by ventricular arrhythmias and sudden death. One form of SQTS is caused by mutation N588K in human ether-a-go-go-related gene (HERG). In this study we sought to determine the potential role of N588K in arrhythmias. METHODS: We measured the characteristics of HERG current generated by wild-type (WT) KCNH2 and the N588K mutant channel expressed in mammalian TSA201 cells. RESULTS: Whole-cell patch-clamp recordings of WT HERG currents showed the usual rapid onset of inactivation (rectification) at potentials more positive than +10 mV. In contrast, N588K currents rectified at potentials over +80 mV. Over the physiological range of potentials, N588K currents do not inactivate. During an action potential clamp, WT currents displayed a "hump" like waveform with slow activation kinetics and a rapid increase during phase 3 repolarization. In contrast, N588K currents were proportional to the amplitude of the action potential and displayed a dome-like configuration and a much larger current during the initial phases in the ventricle. Purkinje cell action potentials display a more negative phase 2 repolarization than the ventricle and elicited much smaller WT and N588K currents of similar amplitudes. CONCLUSIONS: Physiologically the N588K mutation abolishes rectification of HERG currents and specifically increases I(Kr) in the ventricle with minimal effects on the Purkinje fiber action potential duration. Such preferential prolongation may explain the separation of the T and U waves observed in the ECG of SQTS patients and lead to re-excitation of the ventricle endocardium.

Contribution of neuronal sodium channels to the cardiac fast sodium current INa is greater in dog heart Purkinje fibers than in ventricles.

OBJECTIVE: To determine the presence and the potential contribution of neuronal sodium channels to dog cardiac function. METHODS: We used a combination of electrophysiological (patch clamp), RT-PCR, biochemical and immunohistochemical techniques to identify and localize neuronal Na(+) channels in dog heart and determine their potential contribution to the fast sodium current. RESULTS: In all cardiac tissues investigated, Na(v)1.1, Na(v)1.2 and Na(v)1.3 transcripts were detected. In immunoblots, we found Na(v)1.1 and Na(v)1.2 proteins in the ventricle (V) and in Purkinje fibers (PF). Na(v)1.3 immunoblots suggested strong proteolytic activity against this isoform in the heart. Na(v)1.6 was not found in any of the tissues tested. Confocal immunofluorescence on cardiac myocytes showed that Na(v)1.1 was predominantly localized at the intercalated disks in V and PF and around the nucleus (V). Na(v)1.2 was only present at the Z lines (V). Consistent with the immunoblot data, an intense but diffuse intracellular staining was observed for Na(v)1.3. Na(v)1.6 fluorescence staining was faint and diffuse. Surprisingly, immunoblots indicated the presence of two Na(v)beta 2 variants: a 42-kDa protein that co-localized with Na(v)1.2 at the Z lines in V and a 34-kDa protein that co-localized with Na(v)1.1 at the intercalated disks in PF. In agreement with the biochemical data, electrophysiological results suggest that neuronal sodium channels generate 10+/-5% and 22+/-5% of the peak sodium current in dog ventricle and Purkinje fibers, respectively. CONCLUSIONS: Our results suggest that neuronal NaChs are more abundant in Purkinje fibers than in ventricles, and this suggests a role for them in cardiac conduction.

Aluminium-induced impairment of Ca2+ modulatory action on GABA transport in brain cortex nerve terminals.

The gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in vertebrate CNS. At GABAergic synapses, a high-affinity transporter exists, which is responsible for GABA reuptake and release during neurotransmission. GABA transporter activity depends on the phosphorylation/dephosphorylation state, being modulated by Ca(2+)/calmodulin-dependent protein phosphatase 2B (calcineurin). Aluminium is known to interfere with the Ca(2+)/calmodulin signalling pathway. In this work, we investigate the action of aluminium on GABA translocation mediated by the high-affinity transporter, using synaptic plasma membrane (SPM) vesicles and synaptosomes isolated from brain cortex. Aluminium completely relieved Ca(2+) downregulation of GABA transporter, when mediating uptake or release. Accordingly, aluminium inhibited Ca(2+)/calmodulin-dependent calcineurin activity present in SPM, in a concentration-dependent manner. The deleterious action of aluminium on the modulation of GABA transport was ascertained by comparative analysis of the aluminium effect on GABA uptake and release, under conditions favouring SPM dephosphorylation (presence of intracellular micromolar Ca(2+)) or phosphorylation (absence of Ca(2+) and/or presence of W-7, a selective calmodulin antagonist). In conclusion, aluminium-induced relief of Ca(2+) modulatory action on GABA transporter may contribute significantly to modify GABAergic signalling during neurotoxic events in response to aluminium exposure.

Location of the initiation site of calcium transients and sparks in rabbit heart Purkinje cells.

1. The distribution and localization of Ca2+ transients and Ca2+ sparks in isolated adult rabbit Purkinje cells were examined using confocal microscopy and the Ca2+ indicator fluo-3. 2. When cells were field stimulated in 2.0 mM Ca2+ buffer, a transverse confocal line scan (500 Hz) showed that the fluorescence intensity was greatest at the cell periphery during the onset of the Ca2+ transient ([Ca2+]i). In contrast, the [Ca2+]i of ventricular cells showed a more uniform pattern of activation across the cell. Staining with di-8-ANEPPS revealed that Purkinje cells lack t-tubules, whereas ventricular cells have an extensive t-tubular system. 3. When we superfused both cell types with a buffer containing 5 mM Ca2+-1 microM isoproterenol (isoprenaline) they produced Ca2+ sparks spontaneously. Ca2+ sparks occurred only at the periphery of Purkinje cells but occurred throughout ventricular cells. Sparks in both cell types could be completely abolished by addition of the SR inhibitor thapsigargin (500 nM). Brief exposure to nifedipine (10 microM) did not reduce the number of spontaneous sparks. 4. Immunofluorescence staining of Purkinje cells with anti-ryanodine antibody revealed that ryanodine receptors (RyRs) are present at both peripheral and central locations. 5.Computer simulations of experiments in which the calcium transient was evoked by voltage clamp depolarizations suggested that the increase in calcium observed in the centre of the cell could be explained by simple buffered diffusion of calcium. These computations suggested that the RyRs deep within the cell do not contribute significantly to the calcium transient. 6. These results provide the first detailed, spatially resolved data describing Ca2+ transients and Ca2+ sparks in rabbit cardiac Purkinje cells. Both types of events are initiated only at subsarcolemmal SR Ca2+ release sites suggesting that in Purkinje cells, Ca2+ sparks only originate where the sarcolemma and sarcoplasmic reticulum form junctions. The role of the centrally located RyRs remains unclear. It is possible that because of the lack of t-tubules these RyRs do not experience a sufficiently large Ca2+ trigger during excitation-contraction (E-C) coupling to become active.

Early and delayed afterdepolarizations in rabbit heart Purkinje cells viewed by confocal microscopy.

We investigated action potentials and Ca(2+) transients in rabbit Purkinje myocytes using whole cell patch clamp recordings and a confocal microscope. Purkinje cells were loaded with 5 microM Fluo-3/AM for 30min. Action potentials were elicited by application of a stimulus delivered through the recording pipettes. When Purkinje cells were stimulated in 2.0mM Ca(2+), transverse XT line scans revealed a symmetrical 'U'-shaped Ca(2+) transient demonstrating that the transient was initiated at the cell periphery. When Purkinje cells were superfused with 1 microM isoprenaline, both early and delayed afterdepolarizations were induced. XT line scans of cells exhibiting early afterdepolarizations showed a second symmetrical 'U'-shaped transient. This Ca(2+) transient was initiated at the cell periphery suggesting reactivation of the Ca(2+) current. In contrast, in Purkinje cells exhibiting delayed afterdepolarizations and a corresponding transient inward current, XT line scans revealed a heterogenous rise in Ca(2+) at both peripheral and central regions of the cell. Immunofluorescence staining of Purkinje cells with an antibody to ryanodine receptors (RyRs) revealed that RyRs are located at regularly spaced intervals throughout the interior of Purkinje cells. These results suggest that, although RyRs are located throughout Purkinje cells, only peripheral RyRs are activated to produce transients, sparks and early afterdepolarizations. During delayed afterdepolarizations, we observed a heterogenous rise in Ca(2+) at both peripheral and central regions of the cell as well as large central increases in Ca(2+). Although the latter may result from central release, we cannot exclude the possibility that it reflects Ca(2+) diffusion from subsarcolemmal sites.

Ca(2+) regulation of the carrier-mediated gamma-aminobutyric acid release from isolated synaptic plasma membrane vesicles.

The regulation of the carrier-mediated gamma-aminobutyric acid (GABA) efflux was studied in isolated synaptic plasma membrane (SPM) vesicles, which are particularly useful to study neurotransmitter release without interference of the exocytotic machinery. We investigated the effect of micromolar intravesicular Ca(2+) on the GABA release from SPM vesicles under conditions of basal release (superfusion with 150 mM NaCl), homoexchange (superfusion with 500 microM GABA) and K(+) depolarization-induced release (superfusion with 150 mM KCl). We observed that, in the presence of intravesicular Ca(2+) (10 microM), the maximal velocity (J(max)) of K(+) depolarization-induced GABA release is decreased by about 64%, and this effect was abolished in the presence of the channel blocker, La(3+). In contrast, the other mechanisms were not significantly altered by these cations. In agreement with our earlier results, inhibition of GABA uptake by intravesicular Ca(2+) was also observed by determining the kinetic parameters (K(0.5) and J(max)) of influx into the SPM vesicles. Under these conditions, the J(max) of GABA uptake was 17.4 pmol/s per mg protein, whereas in control experiments (absence of Ca(2+)), this value achieved 25.5 pmol/s per mg protein. The inhibitory effect of Ca(2+) on translocation of GABA across SPM appears to be mediated by calcium/calmodulin activation of protein phosphatase 2B (calcineurin), since it was completely relieved by W7 (calmodulin antagonist) and by cyclosporin A (calcineurin inhibitor). These results show that the GABA transport system, operating either in forward or backward directions, requires phosphorylation of internally localized calcineurin-sensitive sites to achieve maximal net translocation velocity.

Modulation of repolarization in rabbit Purkinje and ventricular myocytes coupled by a variable resistance.

Purkinje-ventricular junctions (PVJs) have been implicated as potential sites of arrhythmogenesis, in part because of the dispersion of action potential duration (APD) between Purkinje (P) and ventricular (V) myocytes. To characterize electrotonic modulation of APD as a function of junctional resistance (Rj), we coupled single isolated rabbit P and V myocytes with an electronic circuit. In seven of eight PV myocyte pairs, both APDs shortened on coupling at Rj = 50 MOmega. This was in contrast to modulation of APD in paired ventricular myocytes, which demonstrated APD shortening of the intrinsically longer action potential and APD prolongation of the intrinsically shorter action potential. Companion computer simulations, performed to suggest possible mechanisms for the paradoxical shortening of the V action potential in paired P and V myocytes, showed that the difference in intrinsic peak plateau potentials (Vpp) of the P and V myocytes determined whether the V action potential shortened or prolonged on coupling. This difference in Vpp caused a large, repolarizing coupling current to flow to the V myocyte, contributing to early inactivation of the L-type calcium current and early activation of the inward rectifier current. These results suggest that intrinsic differences in phase 1 repolarization could yield differing patterns of APD shortening or prolongation in the network of subendocardial PVJs, leaving some PVJs vulnerable to conduction of premature stimuli while other PVJs remain refractory.

Identification and properties of ATP-sensitive potassium channels in myocytes from rabbit Purkinje fibres.

OBJECTIVE: Our goal was to identify the ATP-sensitive potassium (KATP) channels in cardiac Purkinje cells and to document the functional properties that might distinguish them from KATP channels in other parts of the heart. METHODS: Single Purkinje cells and ventricular myocytes were isolated from rabbit heart. Standard patch-clamp techniques were used to record action potential waveforms. and whole-cell and single-channel currents. RESULTS: The KATP channel opener levcromakalim (10 microM) caused marked shortening of the Purkinje cell action potential. Under whole-cell voltage-clamp, levcromakalim induced an outward current, which was blocked by glibenclamide (5 microM), in both Purkinje cells and ventricular myocytes. Metabolic poisoning of Purkinje cells with NaCN and 2-deoxyglucose caused a significant shortening of the action potential (control 376 +/- 51 ms; 6 min NaCN/2-deoxyglucose 153 +/- 21 ms). This effect was reversed with the application of glibenclamide. Inside-out membrane patches from Purkinje cells showed unitary current fluctuations which were inhibited by cytoplasmic ATP with an IC50 of 119 microM and a Hill coefficient of 2.1. This reflects approximately five-fold lower sensitivity to ATP inhibition than for KATP channels from ventricular myocytes under the same conditions. The slope conductance of Purkinje cell KATP channels, with symmetric, 140 mM K+, was 60.1 +/- 2.0 pS (mean +/- SEM). Single-channel fluctuations showed mean open and closed times of 3.6 +/- 1.5 ms and 0.41 +/- 0.2 ms, respectively, at -60 mV and approximately 21 degrees C. At positive potentials. KATP channels exhibited weak inward rectification that was dependent on the concentration of internal Mg2+. Computer simulations, based on the above results, predict significant shortening of the Purkinje cell action potential via activation of KATP channels in the range 1-5 mM cytoplasmic ATP. CONCLUSIONS: Purkinje cell KATP channels may represent a molecular isoform distinct from that present in ventricular myocytes. The presence of KATP channels in the Purkinje network suggests that they may have an important influence on cardiac rhythm and conduction during periods of ischemia.

Repolarizing K+ currents in rabbit heart Purkinje cells.

1. Electrophysiological experiments on single myocytes obtained from Purkinje fibres and ventricular tissue of adult rabbit hearts were done to compare the contributions of three potassium (K+) currents to the action potentials in these two tissues. 2. In Purkinje cells reductions in extracellular potassium, [K+]o, from normal (5.4 mM) to 2.0 mM resulted in a large hyperpolarization and marked lengthening of the action potential. In ventricular myocytes, these changes were much less pronounced. Voltage clamp measurements demonstrated that these differences were mainly due to a much smaller inward rectifier K+ current, IK1, in Purkinje cells than in ventricular myocytes. 3. Application of 4-aminopyridine (4-AP, 2 mM) showed that all Purkinje cells exhibited a very substantial Ca2+-independent transient K+ outward current, It. 4-AP significantly broadened the early, rapid repolarization phase of the action potential. 4. Selective inhibitors of the fast component, IK, r (MK-499, 200 nM) and the slow component IK,s (L-735821 (propenamide), 20 nM) of the delayed rectifier K+ currents both significantly lengthened the action potential, suggesting that these conductances are present, but very small (< 20 pA) in Purkinje cells. Attempts to identify time- and voltage-dependent delayed rectifier K+ current(s) in Purkinje cells failed, although a slow delayed rectifier was observed in ventricular myocytes. 5. These results demonstrate significant differences in action potential waveform, and underlying K+ currents in rabbit Purkinje and ventricular myocytes. Purkinje cells express a much smaller IK1, and a larger It than ventricular myocytes. These differences in current densities can explain some of the most important electrophysiological properties of these two tissues.

Conduction between isolated rabbit Purkinje and ventricular myocytes coupled by a variable resistance.

Conduction at the Purkinje-ventricular junction (PVJ) demonstrates unidirectional block under both physiological and pathophysiological conditions. Although this block is typically attributed to multidimensional electrotonic interactions, we examined possible membrane-level contributions using single, isolated rabbit Purkinje (P) and ventricular (V) myocytes coupled by an electronic circuit. When we varied the junctional resistance (Rj) between paired V myocytes, conduction block occurred at lower Rj values during conduction from the smaller to larger myocyte (115 +/- 59 M omega) than from the larger to smaller myocyte (201 +/- 51 M omega). In Purkinje-ventricular myocyte pairs, however, block occurred at lower Rj values during P-to-V conduction (85 +/- 39 M omega) than during V-to-P conduction (912 +/- 175 M omega), although there was little difference in the mean cell size. Companion computer simulations, performed to examine how the early platea currents affected conduction, showed that P-to-V block occurred at lower Rj values when the transient outward current was increased or the calcium current was decreased in the model P cell. These results suggest that intrinsic differences in phase 1 repolarization can contribute to unidirectional block at the PVJ.