Thrombospondin-4 induces prolongation of action potential duration in rat isolated ventricular myocytes.

Expression of thrombospondin-4 (TSP-4), a matricellular protein, is increased in the heart tissue of various cardiac disease models. In dorsal root ganglion neurons, TSP-4 inhibits L-type Ca2+ channel (LTCC) activity. Although TSP-4 might be related to the electrophysiological properties in heart, it remains to be clarified. The present study aimed to clarify the effects of TSP-4 on action potential (AP), LTCC current (ICaL) and voltage-dependent K+ (Kv) channel current (IKv) in rat isolated ventricular myocytes by a patch clamp technique. Ventricular myocytes were isolated from the heart of adult male Wistar rats. The ventricular myocytes were treated with TSP-4 (5 nM) or its vehicle for 4 hr. Then, whole-cell patch clamp technique was performed to measure AP (current-clamp mode) and ICaL and IKv (voltage-clamp mode). The mRNA expression of Kv channels was examined by reverse transcription-polymerase chain reaction. TSP-4 had no effect on the resting membrane potential and peak amplitude of AP. On the other hand, TSP-4 significantly prolonged AP duration (APD) at 50% and 90% repolarization. TSP-4 significantly inhibited the peak amplitudes of ICaL and IKv. TSP-4 had no effect on mRNA expression of Kv channels (Kcna4, Kcna5, Kcnb1, Kcnd2 and Kcnd3). The present study for the first time demonstrated that TSP-4 prolongs APD in rat ventricular myocytes, which is possibly mediated through the suppression of Kv channel activity.

Oxidative status of erythrocytes, hyperglycemia, and hyperlipidemia in diabetic cats.

BACKGROUND: Erythrocytes of diabetic cats have decreased superoxide dismutase activity, possibly indicative of oxidative stress. HYPOTHESIS: Erythrocytes of diabetic cats undergo oxidative stress, which is caused by hyperglycemia and hyperlipidemia, and improves with treatment. ANIMALS: Twenty-seven client-owned cats with diabetes mellitus, 11 matched healthy cats, and 21 purpose-bred healthy cats. METHODS: Prospective study. Advanced oxidized protein products, carbonyls (protein oxidation by-products), and thiols (antioxidants) were quantified in erythrocyte membrane, thiobarbituric acid reactive substances (TBAR, lipid peroxidation by-products), and thiols in erythrocyte cytoplasm of all cats. Comparison were performed between diabetic and matched healthy cats, between diabetic cats achieving remission or not, and among purpose-bred cats after 10 days of hyperglycemia (n = 5) or hyperlipidemia (n = 6) versus controls treated with saline (n = 5) or untreated (n = 5). RESULTS: Compared with controls, erythrocytes of diabetic cats initially had higher median membrane carbonyls (4.6 nmol/mg total protein [range: 0.1-37.7] versus 0.7 [0.1-4.7], P < .001) and lower cytoplasmic TBAR (1.9 nmol/mg [0.5-2.4] versus 2.4 [1.4-3.5] P < .001), and thiols (419 nmol/mg [165-621] versus 633 [353-824], P < 0.001). After 12-16 weeks of treatment in diabetic cats, carbonyls decreased by 13% (P < .001), but remained higher (P < .001) and TBAR and thiols lower (P = .02, P < .001) than those in controls. No differences were observed between diabetic cats achieving remission or not, and among purpose-bred cats. CONCLUSIONS AND CLINICAL IMPORTANCE: Diabetes mellitus is associated with increased protein oxidation and reduced antioxidant defenses, which persist during treatment and remission, although mild improvement in protein oxidation occurs. Short-term hyperglycemia or hyperlipidemia does not cause oxidative stress. The reason for decreased TBAR remains unknown.

Taurine activates glycine and GABAA receptor currents in anoxia-tolerant painted turtle pyramidal neurons.

Unlike anoxia-intolerant mammals, painted turtles can survive extended periods without oxygen. This is partly accomplished by an anoxia-mediated increase in gamma-aminobutyric acid (GABA) release, which activates GABA receptors and mediates spike arrest in turtle neurons via shunting inhibition. Extracellular taurine levels also increase during anoxia; why this occurs is unknown but it is speculated that glycine and/or GABAA/B receptors are involved. Given the general importance of inhibitory neurotransmission in the anoxia-tolerant painted turtle brain, we investigated the function of taurine as an inhibitory neuromodulator in turtle pyramidal neurons. Using whole-cell patch-clamp electrophysiological methods to record from neurons within a cortical brain sheet, we found that taurine depolarized membrane potential by ∼8 mV, increased whole-cell conductance ∼2-fold, and induced an inward current that possessed characteristics similar to GABA- and glycine-evoked currents. These effects were mitigated following glycine receptor antagonism with strychnine and GABAA receptor antagonism with gabazine, bicuculine or picrotoxin, but were unchanged following GABAB or glutamatergic receptor inhibition. These data indicate that a high concentration of taurine in vitro mediates its effects through both glycine and GABAA receptors, and suggests that taurine, in addition to GABA, inhibits neuronal activity during anoxia in the turtle cortex.

Effects of trimethoprim-sulfadiazine and detomidine on the function of equine Kv 11.1 channels in a two-electrode voltage-clamp (TEVC) oocyte model.

The long QT syndrome (LQTS) is a channelopathy that can lead to severe arrhythmia and sudden cardiac death. Pharmacologically induced LQTS is caused by interaction between drugs and potassium channels, especially the Kv 11.1 channel. Due to such interactions, numerous drugs have been withdrawn from the market or are administered with precautions in human medicine. However, some compounds, such as trimethoprim-sulfonamide combinations are still widely used in veterinarian medicine. Therefore, we investigate the effect of trimethoprim-sulfadiazine (TMS), trimethoprim, sulfadiazine, and detomidine on equine-specific Kv 11.1 channels. Kv 11.1 channels cloned from equine hearts were heterologously expressed in Xenopus laevis oocytes, and whole cell currents were measured by two-electrode voltage-clamp before and after drug application. TMS blocked equine Kv 11.1 current with an IC50 of 3.74 mm (95% CI: 2.95-4.73 mm) and affected the kinetics of activation and inactivation. Similar was found for trimethoprim but not for sulfadiazine, suggesting the effect is due to trimethoprim. Detomidine did not affect equine Kv 11.1 current. Thus, equine Kv 11.1 channels are also susceptible to pharmacological block, indicating that some drugs may have the potential to affect repolarization in horse. However, in vivo studies are needed to assess the potential risk of these drugs to induce equine LQTS.

Anesthetic synergy between two n-alkanes.

OBJECTIVE: N-butane and n-pentane can both produce general anesthesia. Both compounds potentiate γ-aminobutyric acid type A (GABAA) receptor function, but only butane inhibits N-methyl-d-aspartate (NMDA) receptors. It was hypothesized that butane and pentane would exhibit anesthetic synergy due to their different actions on ligand-gated ion channels. STUDY DESIGN: Prospective experimental study. ANIMALS: A total of four Xenopus laevis frogs and 43 Sprague-Dawley rats. METHODS: Alkane concentrations for all studies were determined via gas chromatography. Using a Xenopus oocyte expression model, standard two-electrode voltage clamp techniques were used to measure NMDA and GABAA receptor responses in vitro as a function of butane and pentane concentrations relevant to anesthesia. The minimum alveolar concentrations (MAC) of butane and pentane were measured separately in rats, and then pentane MAC was measured during coadministration of 0.25, 0.50 or 0.75 times MAC of butane. An isobole with 95% confidence intervals was constructed using regression analysis. A sum of butane and pentane that was statistically less than the lower-end confidence bound isobole indicated a synergistic interaction. RESULTS: Both butane and pentane dose-dependently potentiated GABAA receptor currents over the study concentration range. Butane dose-dependently inhibited NMDA receptor currents, but pentane did not modulate NMDA receptors. Butane and pentane MAC in rats was 39.4±0.7 and 13.7±0.4 %, respectively. A small but significant (p<0.03) synergistic anesthetic effect with pentane was observed during administration of either 0.50 or 0.75×MAC butane. CONCLUSIONS: Butane and pentane show synergistic anesthetic effects in vivo consistent with their different in vitro receptor effects. CLINICAL RELEVANCE: Findings support the relevance of NMDA receptors in mediating anesthetic actions for some, but not all, inhaled agents.

Assessment of anoxia tolerance and photoperiod dependence of GABAergic polarity in the pond snail Lymnaea stagnalis.

The pond snail Lymnaea stagnalis is reported to be anoxia-tolerant and if the tolerance mechanism is similar to that of the anoxia-tolerant painted turtle, GABA should play an important role. A potentially confounding factor investigating the role of GABA in anoxia tolerance are reports that GABA has both inhibitory and excitatory effects within L. stagnalis central ganglion. We therefore set out to determine if seasonality or photoperiod has an impact on: 1) the anoxia-tolerance of the intact pond snail, and 2) the response of isolated neuroganglia cluster F neurons to exogenous GABA application. L. stagnalis maintained on a natural summer light cycle were unable to survive any period of anoxic exposure, while those maintained on a natural winter light cycle survived a maximum of 4h. Using intracellular sharp electrode recordings from pedal ganglia cluster F neurons we show that there is a photoperiod dependent shift in the response to GABA. Snails exposed to a 16h:8h light:dark cycle in an environmental chamber (induced summer phenotype) exhibited hyperpolarizing inhibitory responses and those exposed to a 8h:16h light:dark cycle (induced winter phenotype) exhibited depolarizing excitatory responses to GABA application. Using gramicidin-perforated patch recordings we also found a photoperiod dependent shift in the reversal potential for GABA. We conclude that the opposing responses of L. stagnalis central neurons to GABA results from a shift in intracellular chloride concentration that is photoperiod dependent and is likely mediated through the relative efficacy of cation chloride co-transporters. Although the physiological ramifications of the photoperiod dependent shift are unknown this work potentially has important implications for the impact of artificial light pollution on animal health.

Canine muscle cell culture and consecutive patch-clamp measurements - a new approach to characterize muscular diseases in dogs.

BACKGROUND: The recognition of functional muscular disorders, (e.g. channelopathies like Myotonia) is rising in veterinary neurology. Morphologic (e.g. histology) and even genetic based studies in these diseases are not able to elucidate the functional pathomechanism. As there is a deficit of knowledge and skills considering this special task, the aim of the current pilot study was to develop a canine muscle cell culture system derived from muscle biopsies of healthy client-owned dogs, which allows sampling of the biopsies under working conditions in the daily veterinary practise. RESULTS: Muscular biopsies from 16 dogs of different age and breed were taken during standard surgical procedures and were stored for one to three days at 4°C in a transport medium in order to simulate shipping conditions. Afterwards biopsies were professionally processed, including harvesting of satellite cells, inducing their proliferation, differentiating them into myotubes and recultivating myotubes after long-term storage in liquid nitrogen. Myogenic origin of cultured cells was determined by immunofluorescence, immunohistology and by their typical morphology after inducing differentiation. Subsequent to the differentiation into myotubes feasibility of patch-clamp recordings of voltage gated ion channels was successfully. CONCLUSION: We have developed a canine muscle cell culture system, which allows sampling of biopsies from young and old dogs of different breeds under practical conditions. Patch clamp measurements can be carried out with the cultured myotubes demonstrating potential of these cells as source for functional research.

In vitro aflatoxin B(1) exposure decreases response to carbamylcholine in the jejunal epithelium of broilers.

The study was conducted to evaluate if aflatoxin B(1) (AFB(1)) has the capacity to affect the electrophysiological variables and active glucose uptake in jejunal epithelium of chicken. For this purpose, intestinal segments from the middle jejunum of broilers (35 to 39 d old) were incubated in Ussing chambers in the presence of 0 (vehicle control), 1.25, 2.50, and 3.75 microg of AFB(1)/mL of buffer. After 40 and 60 min of incubation with AFB(1), d-glucose (20 mmol/L) and carbamylcholine (200 micromol/L; an analog of acetylcholine and inducer of apical Cl(-) secretion) were respectively added to the incubation medium. Addition of 3.75 microg of AFB(1) caused an increase (P < 0.04) in short-circuit current (I(sc)) and transmural potential difference (V(t)) between 12 to 27 min postexposure as compared with the control. Glucose-induced DeltaI(sc) and percentage of DeltaV(t) were reduced (P < 0.04) at 2.5 and 3.75 microg of AFB(1)/mL, respectively, as compared with the control. The carbamylcholine-induced DeltaI(sc) and DeltaV(t) were both lower (P < 0.05) at 3.75 microg of AFB(1)/mL as compared with the control (-0.05 microA/cm(2), 0.1 mV vs. 1.1 microA/cm(2), and 0.6 mV, respectively). These observations indicate that acute exposure to AFB(1) may increase apical anion secretion in the jejunal epithelium of chicken. The negative effect of this increased anion secretion on active glucose uptake was, however, not prominent and may be considered as moderate or progressive in nature.

The patch clamp technique: principles and technical considerations.

The development of techniques that allow the high fidelity measurement of small scale ionic currents ushered in a new era of investigation into the role of ion channels in the physiologic and pathophysiologic function of excitable tissue. Based upon the formation of a high resistance (gigaohm) seal with the membrane of the cell being studied, these "patch clamp" techniques have improved our understanding of a wide variety of cardiac disease states with respect to diagnosis, treatment and prognosis. This review outlines the basic principles underlying the patch clamp technique, including the properties of biological membranes and ion channels, and provides an elementary summary of its application to the recording of cardiac ionic currents, with a particular focus on issues related to myocyte isolation, electrode manufacturing and the voltage clamp configuration.

Voltage-gated currents of tilapia prolactin cells.

The first recordings of neuron-like electrical activity from endocrine cells were made from fish pituitary cells. However, patch-clamping studies have predominantly utilized mammalian preparations. This study used whole-cell patch-clamping to characterize voltage-gated ionic currents of anterior pituitary cells of Oreochromis mossambicus in primary culture. Due to their importance for control of hormone secretion we emphasize analysis of calcium currents (I(Ca)), including using peptide toxins diagnostic for mammalian neuronal Ca(2+) channel types. These appear not to have been previously tested on fish endocrine cells. In balanced salines, inward currents consisted of a rapid TTX-sensitive sodium current and a smaller, slower I(Ca); there followed outward potassium currents dominated by delayed, sustained TEA-sensitive K(+) current. About half of cells tested from a holding potential (V(h)) of -90 mV showed early transient K(+) current; most cells showed a small Ca(2+)-mediated outward current. I-V plots of isolated I(Ca) with 15 mM [Ca(2+)](o) showed peak currents (up to 20 pA/pF from V(h) -90 mV) at approximately +10 mV, with approximately 60% I(Ca) for V(h) -50 mV and approximately 30% remaining at V(h) -30 mV. Plots of normalized conductance vs. voltage at several V(h)s were nearly superimposable. Well-sustained I(Ca) with predominantly Ca(2+)-dependent inactivation and inhibition of approximately 30% of total I(Ca) by nifedipine or nimodipine suggests participation of L-type channels. Each of the peptide toxins (omega-conotoxin GVIA, omega-agatoxin IVA, SNX482) alone blocked 36-54% of I(Ca). Inhibition by any of these toxins was additive to inhibition by nifedipine. Combinations of the toxins failed to produce additive effects. I(Ca) of up to 30% of total remained with any combination of inhibitors, but 0.1mM cadmium blocked all I(Ca) rapidly and reversibly. We did not find differences among cells of differing size and hormone content. Thus, I(Ca) is carried by high voltage-activated Ca(2+) channels of at least three types, but the molecular types may differ from those characterized from mammalian neurons.

Pharmacological profile of inhibition of the chloride channels activated by extracellular acid in cultured rat Sertoli cells.

Sertoli cells from mammalian testis are key cells involved in the development and maintenance of stem cell spermatogonia as well as in the secretion of a Cl(-) and K(+)-rich fluid into the lumen of seminiferous tubules. The pharmacology and contribution of Cl(-) channels to the physiology of Sertoli cells were investigated using whole-cell patch clamp and iodide efflux experiments applied to cultured rat Sertoli cells. We characterized an outwardly rectifying Cl(-) current stimulated by various acid species including the physiologically relevant lactic acid. Using the iodide efflux technique, the pharmacological properties of this Cl(-) current, noted ICl(acid), revealed Ca(2+)-independent inhibition by DIDS (IC(50) = 27 microM), glibenclamide (IC(50) = 31 microM) and DPC (IC(50) = 86 microM). ICl(acid) was neither affected by calix[4]arene nor by 9-AC. The order of potency for inhibition of ICl(acid) is DIDS approximately glibenclamide > DPC >> calix[4]arene, 9-AC. For comparison, the inhibitory profile of the swelling- and ATP-activated Cl(-) currents in Sertoli cells is DPC = DIDS >> glibenclamide = 9-AC for ICl(swell) and DPC = 9-AC = DIDS >> glibenclamide for ICl(ATP). This description provides new insights into the physiology and pharmacology of the endogenous Cl(-) channels expressed and potentially involved in fluid secretion in Sertoli cells.

Single-channel response of hamster oocytes to fertilization with homologous spermatozoa

Electrophysiological events occur early after fertilization, along with changes in intracellular Ca2+ concentration. Passive electrical parameters were determined in golden hamster oocytes by whole cell patch-clamp method. In separate experiments the effect of 4-aminopyridine on resting oocytes was tested. The single-channel patch clamp configuration was employed to assess the electrical response to fertilization with homologous sperm. Structure of oocytes submitted to patch clamp was evaluated with scanning electron microscopy and found to be preserved.Oocyte diameter was 70.2 ± 2.2 µm; their resting parameters were: membrane potential 23.8 ± 0.8 mV; total membrane specific resistance 519.1 ± 94.6 Ù.cm2, and specific capacity 0.99 ± 0.03 µF.cm-2. Total membrane current was decreased by 42 % by 4-aminopyridine.Control oocytes and oocytes exposed to sperm differed in their membrane currents in response to a voltage ramp clamping membrane potential from - 100 mV to + 100 mV. In both cases, currents were largest at the most negative potentials, but sperm-exposed oocytes had larger currents. Additionally, while in controloocytes the current was inward at negative potentials but outward at positive potentials, in the presence of spermatozoa oocytes was inward within the whole voltage range tested. This latter current may represent Ca2+ en try

Salmonella enterica serovar Choleraesuis infection of the porcine jejunal Peyer's patch rapidly induces IL-1beta and IL-8 expression.

Salmonella enterica serovar Choleraesuis is an enteric pathogen of swine, producing septicemia, enterocolitis, pneumonia, and hepatitis. The initial molecular events at the site of Salmonella infection are hypothesized to be critical in the initiation of innate and adaptive immune responses; however, the acute immune response elicited by porcine intestinal tissues is not well understood. To address this need, we employed explants of jejunal Peyer's patch (JPP) mucosa from pigs to examine Salmonella-induced immune responses under controlled conditions as well as to overcome limitations of whole animal approaches. JPP explants mounted in Ussing chambers maintained normal histological structure for 2 h and stable short-circuit current and electrical conductance for 2.5 h. After ex vivo luminal exposure to Salmonella serovar Choleraesuis, JPP responded with an increase in mRNA expression of IL-1beta and IL-8, but not TNFalpha. Increased IL-1beta and IL-8 expression were dependent on efficient Salmonella adhesion and internalization, whereas mutant Salmonella did not induce inflammatory cytokine expression. Commensal enteric bacteria, present in some experiments, also did not induce inflammatory cytokine expression. These findings indicate that Salmonella uptake by Peyer's patch is important in the induction of an innate response involving expression of IL-1beta and IL-8, and that ex vivo intestinal immune tissue explants provide an intact tissue model that will facilitate investigation of mucosal immunity in swine.

Drug resistance and neurotransmitter receptors of nematodes: recent studies on the mode of action of levamisole.

Here we review recent studies on the mode of action of the cholinergic anthelmintics (levamisole, pyrantel etc.). We also include material from studies on the free living nematode Caenorhabditis elegans. The initial notion that these drugs act on a single receptor population, while attractive, has proven to be an oversimplification. In both free living and parasitic nematodes there are multiple types of nicotinic acetylcholine receptor (nAChR) on the somatic musculature. Each type has different (sometimes subtly so) pharmacological properties. The implications of these findings are: (1) combinations of anthelmintic that preferentially activate a broad range of nAChR types would be predicted to be more effective; (2) in resistant isolates of parasite where a subtype has been lost, other cholinergic anthelmintics may remain effective. Not only are there multiple types of nAChR, but relatively recent research has shown these receptors can be modulated; it is possible to increase the response of a parasite to a fixed concentration of drug by altering the receptor properties (e.g. phosphorylation state). These findings offer a potential means of increasing efficacy of existing compounds as an alternative to the costly and time consuming development of new anthelmintic agents.

Electrophysiological investigation of anthelmintic resistance.

It is pointed out that two of the three major groups of anthelmintic act by opening membrane ion-channels. It is appropriate, therefore, to use electrophysiological methods to study the properties of the sites of action of these drugs and the changes in the properties of these receptor sites associated with resistance. This paper describes the use of the patch-clamp technique to observe the currents that flow through the levamisole-activated channels as they open and close in levamisole-sensitive and levamisole-resistant isolates. It was found that, on average, the proportion of time the channels are open, is less in the resistant isolate. The patch-clamp technique also showed that the ion-channels are heterogeneous and that one of the subtypes is lost with the appearance of resistance. The use of the current clamp technique is illustrated to record a site of action of ivermectin in the pharyngeal muscle of Ascaris.