Effects of black widow spider venom and Ca2+ on quantal secretion at the frog neuromuscular junction

A modification of the classical procedure of fluctuation analysis is used to measure the waveform, w(t), mean amplitude, (h), and mean rate of occurrence, (r), of miniature endplate potentials (MEPPs) at frog cutaneous pectoris neuromuscular junctions treated with black widow spider venom (BWSV). MEPP parameters are determined from the power spectrum of the fluctuating potential and the second (variance), third (skew), and fourth semi-invariants (cumulants) of high-pass-filtered records of the potential. The method gives valid results even when the mean potential undergoes slow changes unrelated to MEPPs and when the MEPP rate is not stationary; it detects changes in the distribution of MEPP amplitudes and corrects for the nonlinear summation of MEPPs. The effects of Ca2+ on BWSV-induced secretion are studied in detail. When Ca2+ is absent, the power spectrum of the fluctuations is shaped like the spectrum of w(t) and secretion is quasi-stationary; (r) rises smoothly to peak values of approximately 1,500/s and then quickly subsides to levels near 10/s. Many relatively small and some "giant" MEPPs occur at the ends of the experiments, and the distribution of MEPP amplitudes broadens. When the effects of this broadening are corrected for, we find that approximately 0.7 X 10(6) MEPPs occurred during the 30 min of intense secretion. Since BWSV depletes nerve terminals of their quanta of transmitter and their synaptic vesicles, this figure is an upper limit for the quantal store in a resting terminal. When Ca2+ is present, the noise spectrum deviates from the spectrum of w(t) and secretion is nonstationary; (r) rises to similar peak values but is sustained at levels near 400/s for up to an hour and at least 1.5 X 10(6) quanta are secreted within this period. Thus, the quantal store must have turned over at least twice under this condition. Data previously obtained at junctions treated with La3+ are corrected for nonlinear summation and for the distribution of MEPP amplitudes. The two corrections roughly compensate each other, and the corrected results confirm the previous conclusion that the number of quanta secreted from La3+-treated terminals during 1 h is not strongly dependent upon the extracellular concentration of Ca2+; approximately 2 X 10(6) quanta are released even when Ca2+ is absent.     

Assessment of binding of L-cystine and L-lysine by rat renal brush-border membranes

Cystine and lysine bind to isolated rat renal brush-border vesicles. Three methods to determine the extent of amino acid binding to the membranes have been compared, one relying on the osmotic reactivity of the vesicle, a second by trichloroacetic acid precipitation of membrane-bound material and a third by initial rate analysis. For cystine, all methods yield comparable results at early time points, indicating the trichloroacetic acid method is a simple and valuable tool for binding estimation under initial-rate or near initial-rate conditions. For lysine, initial rate analysis and osmotic perturbation are the methods of choice since lysine co-precipitates with trichloroacetic acid.     

Cation chelators and their utilization in the preparation of low concentrations of calcium. Caution of use in biological systems with high affinity to calcium

Ethylene glycol bis (beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA)-calcium buffer is widely used in various calcium-dependent reactions where free calcium concentrations of 1 microM or less are desirable. The free calcium concentration is calculated from the association constant of EGTA . Ca2- and serves as the true available calcium in systems devoid of a constituent with high affinity to Ca2+ other than EGTA. But, it is conceivable that in systems with high affinity to Ca2+ (comparable to that of EGTA) this is not the case, because such systems will compete with EGTA for the total calcium content in the medium, so that the true available calcium for these systems is greater than that calculated from the EGTA buffer. This hypothesis was tested in three different Ca+-modulated systems: Quin 2 fluorescence, Ca2+-ATPase, and adenylate cyclase, in which the response of the system to calcium was compared between EGTA-free media, containing known amounts of added calcium, and the EGTA-Ca2+ buffer media. In all three systems, the amount of available calcium in the EGTA-Ca2+ buffer medium was much greater than the calculated free Ca2+ concentration. This indicates that in systems with high affinity to Ca2+, preparation of available Ca2+ in concentrations of 1 microM or less must account for both the EGTA and the system capacities for calcium.     

Transport and metabolism of galactose in rat kidney cortex

1. Analysis of transport of d-galactose was complicated by metabolism of the compound but appeared to have two components: a substrate-saturable component and a diffusion component. At low substrate concentration (<1mm) active transport was observed. Accumulation of galactose was largely independent of Na(+) concentration. The apparent K(m) for this component was 0.2mm. At substrate concentrations above 1mm the active transport system appeared saturated and further increases in substrate concentration resulted in a linear increase in the rate of galactose accumulation, but no concentration gradient was formed. 2. d-[1-(14)C]Galactose (2mm) was metabolized to (14)CO(2) by rat kidney-cortex slices incubated at 37 degrees C, at the rate of 68nmol/h per 100mg of tissue. 3. Intracellular components from such incubations were separated into a neutral fraction, the only major labelled component being galactose, and a phosphorylated fraction. 4. Phosphorylated metabolites found in galactose-incubated slices increased with increasing substrate concentration and achieved a limiting value of 0.42mm after 60min of incubation. 5. Galactose uptake was inhibited by anaerobiosis, dinitrophenol and phlorrhizin. 6. Methyl alpha-d-glucoside and d-glucose partially inhibited galactose uptake only at ratios of 100:1. 7. The presence of pyruvate did not decrease galactose metabolism although it did decrease production of (14)CO(2) from [1-(14)C]galactose. Gluconeogenesis occurred in the presence of pyruvate and (14)C from galactose was found in glucose. 8. Rat kidney-cortex slices metabolized 2mm-[1-(14)C]galactonate to (14)CO(2) at a rate of 20nmol/h per 100mg of tissue.     

Expression of rat jejunal cystine carrier in Xenopus oocytes

Functional interactive cystine-lysine carriers have been expressed in Xenopus oocytes following the injection of RNA extracted from rat intestinal mucosa. Lysine-inhibitable cystine uptake was able to be measured 16 h after oocyte injection with RNA. The longer the oocytes were maintained after injection, the more cystine transport capability was induced. Uninjected or water-injected oocytes showed virtually no lysine-inhibitable cystine uptake, and no system developed after the oocytes had been isolated and maintained in vitro. The cystine uptake expressed after RNA injection was selectively inhibited by dibasic amino acids and phenylalanine but not by other amino acids or alpha-methyl-D-glucoside. Expression of the interactive cystine-lysine system was induced only by RNA isolated from intestinal tissue and not by RNA from rat liver. The Km for cystine uptake in RNA-injected oocytes was 0.01 mM and appears identical to the single system found in the RNA source tissue.     

Atopic disease and immunoglobulin E in twins reared apart and together.

Both genetic and environmental influences have been implicated in the etiology of atopic disease and in the determination of serum IgE levels. To quantify the relative contribution of these influences, we studied the prevalence of asthma and seasonal rhinitis, skin-test response, total serum IgE levels, and specific IgE, as measured by RAST, in a sample of MZ and DZ twins reared apart or together. Concordance rates for asthma, rhinitis, positive skin tests, and RAST were calculated. MZ twins, whether reared apart or together, showed a greater concordance than dizygotic twins reared apart or together. Maximum-likelihood tests of genetic and environmental components of the variation of total IgE levels revealed a substantial genetic component and a negligible contribution from common familial environmental effects.