Biomedical subjects
Publications and source records attributed to M D Leibowitz.
The design and construction of a novel apparatus to fire polish patch-clamp recording pipets is described. The device positions the polishing filament in the field of view of the high-power polishing objective and uses the mechanical stage of the microscope to hold the electrode, eliminating the need for a micromanipulator to hold either the filament or electrode.
Membrane patches were excised from enzymatically dissociated frog toe muscle. High-conductance anion channels could be induced in previously quiet patches by 20-120 s depolarizations beyond +20 mV and then studied in the potential range from -80 to +60 mV for a long time. From reversal potentials the estimated permeability ratios PCl/PNa and PCl/Pglucuronate were near 3.5 and 4, respectively. There were probably 5 or more conductance levels (substates) for a single channel, the most common in symmetrical 110 mM NaCl being 260 and 70 pS at 10 degrees C. Gating was complex, with rapid and slow events and several gating modes, including periods of rapid flickering. Channels closed reversibly at potentials more negative than -50 mV. The channel was blocked by application to the cytoplasmic face of tannic acid, gallic acid, and zinc but not of DIDS or 9-anthracene-carboxylic acid, and it was blocked by extracellular zinc.
Macroscopic currents in Na channels were recorded from adult frog skeletal muscle under voltage clamp as various toxins were added to the bathing medium. Veratridine, cevadine, and 3-(4-ethoxybenzoyl)-veracevine modified the Na channels in a use-dependent manner during depolarizations and held them open for 3, 2.4, and 1.2 s, respectively, at -90 mV. The three alkaloids modified channels in the same way. Activation gating was shifted about -100 mV by the modification, and reversible closing of the channels by strong hyperpolarizations slowed reversal of the modification. The synthetic insecticides deltamethrin, EDO, GH739, and GH414 also modified channels during depolarizations that opened channels. The modification lasted 3 s with deltamethrin, but only 3-5 ms with the others. Hyperpolarization speeded the shutting off of current in insecticide-modified channels, but no reversible activation gating could be demonstrated. The ionic selectivity, PNa/PNH4, of channels was decreased by all of the toxins. This ratio was 0.11 in normal channels, 0.26 in insecticide-modified channels, and 0.7-1.6 in veratrum-alkaloid-modified channels. During use-dependent modification, the veratrum alkaloids reduced the total Na current markedly, while deltamethrin did not. Thus, alkaloid and insecticide modifications share many features but differ in how much the conducting properties of the pore are changed and whether the channel can close reversibly while the toxin remains bound.
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Na channels of frog muscle fibers treated with 100 microM veratridine became transiently modified after a train of repetitive depolarizations. They open and close reversibly with a gating process whose midpoint lies 93 mV more negative than the midpoint of normal activation gating and whose time course shows no appreciable delay in the opening or closing kinetics but still requires more than two kinetic states. Like normal activation, the voltage dependence of the modified gating can be shifted by changing the bathing Ca2+ concentration. The instantaneous current-voltage relation of veratridine-modified channels is curved at potentials negative to -90 mV, as if external Ca ions produced a voltage-dependent block but also permeated. Modified channels probably carry less current than normal ones. When the concentration of veratridine is varied between 5 and 100 microM, the initial rate of modification during a pulse train is directly proportional to the concentration, while the rate of recovery from modification after the train is unaffected. These are the properties expected if drug binding and modification of channels can be equated. Hyperpolarizations that close modified channels slow unbinding. Allethrin and DDT also modify channels. They bind and unbind far faster than veratridine does, and their binding requires open channels.
The temporal relationships among junctional acetylcholine receptor single-channel currents have been examined to probe the mechanism of channel activation. We have presented an analytical approach, termed single-channel ensemble analysis, that allows one to estimate the kinetic transition rate constants for channel-opening and closing as well as the rate of leaving the specific doubly-liganded, closed state from which opening occurs. This approach may be applied to data produced by any number of independent channels as long as the probability of channel opening is low, a condition that is experimentally verifiable. The method has been independently validated using simulated single-channel data generated by computer from one or 100 hypothetical channels. Typical experimental values for the transition rate constants estimated from acetylcholine-activated single channels at the garter snake neuromuscular junction were: opening = 1,200 s-1, closing = 455 s-1, back rate for leaving the doubly-liganded, closed state = 3,200 s-1 at a transmembrane potential of -92 mV at room temperature. Each of these three rate constants was voltage dependent, with the closing rate decreasing e-fold for 173 mV of hyperpolarization, the opening rate increasing e-fold for 78 mV, and the unbinding rate increasing e-fold for 105 mV. The channel-closing rate was agonist dependent, being greater at all potentials for channels activated with carbamylcholine than for channels activated with acetylcholine. However, the single-channel conductance and reversal potential were the same for these two agonists.
Single-channel currents from acetylcholine receptor channels of garter snake neuromuscular junctions were recorded using the patch-clamp technique. Low concentrations of acetylcholine or carbamylcholine induced populations of single current events whose amplitudes and durations had unimodal distributions. The probability with which channel opening transitions occurred was time dependent, so that it was more probable for channels to open during the several hundred microseconds following a closing transition than during any later equivalent interval. The time-dependent distributions of duration and opening-transition probability were fitted by a sequential, reversible kinetic model in which the agonist binding steps occur before, and separately from, channel activation. This description allowed estimates to be obtained of both the opening (approximately 750s-1) and closing (approximately 500s-1) transition rates of these channels and of the mean lifetimes of the open- (approximately 2 ms) and the closed-channel state (approximately 200 mus) to which the open state was reversibly related.
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UNLABELLED: The peroxisome proliferator-activated receptor (PPAR) gamma is highly expressed in the colon mucosa. In vitro, it regulates inflammation. AIM: To evaluate the anti-inflammatory functions of PPARgamma agonist during a trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice. METHODS: Colitis was induced in Balb/c mice after intra-rectal administration of TNBS. The intensity of inflammation was assessed 2 and 5 days after colitis induction by macroscopic and histologic scores and by the quantification of colon myeloperoxidase (MPO), IL-1B and TNFalpha mRNA concentrations. The therapeutic role of PPARgamma agonist given by oral gavage was assessed in preventive and treatment modes. RESULTS: TNBS induced severe macroscopic and histologic lesions, with high mucosal MPO, IL-1B and TNFalpha mRNA concentrations. PPARgamma agonist given preventively or in treatment mode allowed a significant decrease of macroscopic and histologic scores through a normalization of MPO, IL-1B and TNFalpha mRNA concentrations. CONCLUSION: PPARgamma agonist decreases the intensity of TNBS induced colitis through normalization of IL-1B and TNFalpha expression. PPARgamma agonists may be proposed as new therapeutic agents in inflammatory bowel diseases.