Effects of electro-mechanical uncouplers, hormonal stimulation and pacing rate on the stability and function of cultured rabbit myocardial slices.

Introduction: Recent advances have enabled organotypic culture of beating human myocardial slices that are stable for weeks. However, human myocardial samples are rare, exhibit high variability and frequently originate from diseased hearts. Thus, there is a need to adapt long-term slice culture for animal myocardium. When applied to animal cardiac slices, studies in healthy or genetically modified myocardium will be possible. We present the culture of slices from rabbit hearts, which resemble the human heart in microstructure, electrophysiology and excitation-contraction coupling. Methods: Left ventricular myocardium from New Zealand White rabbits was cut using a vibratome and cultured in biomimetic chambers for up to 7 days (d). Electro-mechanical uncoupling agents 2,3-butanedione monoxime (BDM) and cytochalasin D (CytoD) were added during initiation of culture and effects on myocyte survival were quantified. We investigated pacing rates (0.5 Hz, 1 Hz, and 2 Hz) and hormonal supplements (cortisol, T3, catecholamines) at physiological plasma concentrations. T3 was buffered using BSA. Contractile force was recorded continuously. Glucose consumption and lactate production were measured. Whole-slice Ca2+ transients and action potentials were recorded. Effects of culture on microstructure were investigated with confocal microscopy and image analysis. Results: Protocols for human myocardial culture resulted in sustained contracture and myocyte death in rabbit slices within 24 h, which could be prevented by transient application of a combination of BDM and CytoD. Cortisol stabilized contraction amplitude and kinetics in culture. T3 and catecholaminergic stimulation did not further improve stability. T3 and higher pacing rates increased metabolic rate and lactate production. T3 stabilized the response to ß-adrenergic stimulation over 7 d. Pacing rates above 1 Hz resulted in progredient decline in contraction force. Image analysis revealed no changes in volume fractions of cardiomyocytes or measures of fibrosis over 7 d. Ca2+ transient amplitudes and responsiveness to isoprenaline were comparable after 1 d and 7 d, while Ca2+ transient duration was prolonged after 7 d in culture. Conclusions: A workflow for rabbit myocardial culture has been established, preserving function for up to 7 d. This research underscores the importance of glucocorticoid signaling in maintaining tissue function and extending culture duration. Furthermore, BDM and CytoD appear to protect from tissue damage during the initiation phase of tissue culture.

Channel characteristics of VDAC-3 from Arabidopsis thaliana.

Four different isoforms of the Voltage-Dependent Anion Channel (VDAC) have been identified in Arabidopsis plant cells. The electrophysiological characteristics of several VDAC channels from animal as well as plant cells are well documented, but those of this model plant are unknown. One isoform, AtVDAC-3 was obtained either directly by cell-free synthesis or produced in Escherichia coli, as inclusion bodies, and re-natured. An electrophysiological study of the purified proteins in planar lipid bilayers showed that both methods yielded proteins with similar channel activity. The characteristics of AtVDAC-3 are that of a bona fide VDAC-like channel.

Relative contribution of SKCa and TREK1 channels in purinergic and nitrergic neuromuscular transmission in the rat colon.

Purinergic and nitrergic neurotransmission predominantly mediate inhibitory neuromuscular transmission in the rat colon. We studied the sensitivity of both purinergic and nitrergic pathways to spadin, a TWIK-related potassium channel 1 (TREK1) inhibitor, apamin, a small-conductance calcium-activated potassium channel blocker and 1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ), a specific inhibitor of soluble guanylate cyclase. TREK1 expression was detected by RT-PCR in the rat colon. Patch-clamp experiments were performed on cells expressing hTREK1 channels. Spadin (1 µM) reduced currents 1) in basal conditions 2) activated by stretch, and 3) with arachidonic acid (AA; 10 µM). l-Methionine (1 mM) or l-cysteine (1 mM) did not modify currents activated by AA. Microelectrode and muscle bath studies were performed on rat colon samples. l-Methionine (2 mM), apamin (1 µM), ODQ (10 µM), and N(ω)-nitro-l-arginine (l-NNA; 1 mM) depolarized smooth muscle cells and increased motility. These effects were not observed with spadin (1 µM). Purinergic and nitrergic inhibitory junction potentials (IJP) were studied by incubating the tissue with l-NNA (1 mM) or MRS2500 (1 µM). Both purinergic and nitrergic IJP were unaffected by spadin. Apamin reduced both IJP with a different potency and maximal effect for each. ODQ concentration dependently abolished nitrergic IJP without affecting purinergic IJP. Similar effects were observed in hyperpolarizations induced by sodium nitroprusside (1 µM) and nitrergic relaxations induced by electrical stimulation. We propose a pharmacological approach to characterize the pathways and function of purinergic and nitrergic neurotransmission. Nitrergic neurotransmission, which is mediated by cyclic guanosine monophosphate, is insensitive to spadin, an effective TREK1 channel inhibitor. Both purinergic and nitrergic neurotransmission are inhibited by apamin but with different relative sensitivity.

On the specificity of pig adrenal ferredoxin (adrenodoxin) and spinach ferredoxin in electron-transfer reactions.

Spinach leaf ferredoxin and ferredoxin:NADP oxidoreductase as well as pig adrenodoxin and adrenodoxin reductase have been purified to homogeneity. Ferredoxin-NADP reductase and adrenodoxin-NADP reductase can perform the same diaphorase reactions (dichloroindophenol, ferricyanide and cytochrome c reduction) albeit not with the same efficiency. Despite the differences in their redox potentials, animal and plant ferredoxins can be used as heterologous substrates by the ferredoxin-NADP reductases from both sources. In heterologous systems, however, the ferredoxin/adrenodoxin concentrations must be increased approximately 100-fold in order to reach rates similar to those obtained in homologous systems. Ferredoxin and adrenodoxin can form complexes with the heterologous reductases as demonstrated by binding experiments on ferredoxin-Sepharose or ferredoxin-NADP-reductase-Sepharose and by the realization of difference spectra. Adrenodoxin also weakly substitutes for ferredoxin in NADP photoreduction, and can be used as an electron carrier in the light activation of the chloroplastic enzyme NADP-dependent malate dehydrogenase. In addition adrenodoxin is a good catalyst of pseudocyclic photophosphorylation, but not of cyclic phosphorylation and can serve as a substrate of glutamate synthase. These results are discussed with respect to the known structures of plant and animals ferredoxins and their respective reductases.

[The diagnosis of Gougerot-Sjorgen syndrome in rheumatology. I. Evaul ation of the principal complementary examinations]. / Diagnostic du syndrome de Gougerot-Sjögren en rhumatologie. I. Evaluation des principaux examens complémentaires.

The examination of a patient with Sjögren's syndrome includes evaluation of the eye, the buccal cavity, and a search for certain factors in the blood. Schirmer's blotting-paper test is a good test but is not specific. In addition, a decreased amount of tearing is difficult to interpret after the age of 45. Slit-lamp examination (rose bengal and fluorescein) yields lesions which confirm keratoconjunctivitis due to decreased tearing. The buccal component is difficult to evaluate. A biopsy of the buccal mucosa gives the best results with minimum risk and expense. Nucleotide scanning is sensitive, but less specific. Salivary flow decreases with age. After 60 years of age this decrease can not be interpreted. The chemical composition of tears or of saliva is promising, but it is not yet a part of the usual diagnostic work-up. Of the available laboratory tests, anti-SS-A antibodies and/ or anti-SS-B antibodies are of value, but they are not found consistently.

Secondary foci of primary tumors of the bladder in the upper urinary tract.

In 10 years the authors have observed 16 cases of upper urinary tract tumors, appearing from 2 to 16 years after the initial diagnosis of bladder carcinoma. Such lesions are more frequent than has been suggested in the literature and represent 25% of all tumors of the renal pelvis and ureter seen in our service during this period. We have shown vesicoureteral reflux directly in 6 cases, indirectly in a further 6 with suggestive signs in 3 more. We believe that reflux causing the implantation of desquamated tumor cells from the bladder tumor is the most important pathogenetic mechanism for upper urinary tract "recurrences." The interval between the initial diagnosis of bladder tumor and the appearance of secondary foci may be longer than 20 years. Prolonged surveillance is therefore necessary, particularly if vesicoureteral reflux has been shown.