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Notes: Abstract: Methylmercury (MeHg) increases the concentration of intracellular Ca2+ ([Ca2+]i) and another endogenous polyvalent cation in both synaptosomes and NG108-15 cells. In synaptosomes, the elevation in [Ca2+]i was strictly dependent on extracellular Ca2+ (Ca2+e); similarly, in NG108-15 cells, a component of the elevations in [Ca2+]i was Ca2+e dependent. The MeHg-induced elevations in endogenous polyvalent cation concentration were independent of Ca2+e in synaptosomes and NG108-15 cells. The pattern of alterations in fura-2 fluorescence suggested the endogenous polyvalent cation may be Zn2+. Using 19F-NMR spectroscopy of rat cortical synaptosomes loaded with the fluorinated chelator 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid (5F-BAPTA), we have determined unambiguously that MeHg increases the free intrasynaptosomal Zn2+ concentration ([Zn2+]i). In buffer containing 200 µM EGTA to prevent the Ca2+e-dependent elevations in [Ca2+]i, the [Zn2+]i was 1.37 ± 0.20 nM; following a 40-min exposure to MeHg-free buffer [Zn2+]i was 1.88 ± 0.53 nM. Treatment of synaptosomes for 40 min with 125 µM MeHg yielded [Zn2+]i of 2.69 ± 0.55 nM, whereas 250 µM MeHg significantly elevated [Zn2+]i to 3.99 ± 0.68 nM. No Zn2+ peak was observed in synaptosomes treated with the cell-permeant heavy metal chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN, 100 µM) following 250 µM MeHg exposure. [Ca2+]i in buffer containing 200 µM EGTA was 338 ± 26 nM and was 370 ± 64 nM following an additional 40-min exposure to MeHg-free buffer. [Ca2+]i was 498 ± 28 or 492 ± 53 nM during a 40-min exposure to 125 or 250 µM MeHg, respectively. None of the values of [Ca2+]i differed significantly from either pretreatment levels or buffer-treated controls.

Notes: Abstract: Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune neuromuscular disease in which impairment of Ca2+ entry into the nerve ending and consequent impaired release of acetylcholine (ACh) results in muscle weakness. The identity of the primary antigenic target molecule(s) of the autoantibodies is uncertain. Electrophysiological studies and 45Ca2+ uptake studies implicate a direct effect on the Ca2+ channel complex at the motor nerve terminal. Some recent studies, however, suggest a more indirect interference caused by binding of autoantibodies to synaptotagmin or syntaxin, molecules presumed to be involved in docking and/or coupling the synaptic vesicles to the Ca2+ channels in the active zone for vesicle exocytosis and transmitter release. Western blot analyses of rat and human brain membrane proteins and pure recombinant synaptotagmin and syntaxin were used to examine directly the targets of LEMS autoantibodies and determine specifically whether or not synaptotagmin and/or syntaxin were general targets in LEMS. IgG from 14 patients with LEMS was used to probe western blots of gels containing synaptotagmin, syntaxin, rat synaptosomal proteins, and human brain membrane proteins. Several similar immunoreactive bands were observed using both rat and human brain membranes. These include high-molecular-weight protein bands whose size would be consistent with being components of Ca2+ channels. No reactive component was observed against either syntaxin or synaptotagmin in IgG of the 14 LEMS patients. However, both human and rat brain membranes contain proteins recognized by antibodies directed against synaptotagmin or syntaxin, indicating their immunologic relatedness and evolutionary conservation. These results suggest that large-molecular-weight proteins consistent with being Ca2+ channel subunits rather than syntaxin and synaptotagmin are general targets of LEMS autoantibodies.

Notes: Abstract: The cationic potentiometric fluorescent probe 3,3′-diethylthiadicarbocyanine iodide [DiS-C2(5)] was used in synaptosomes to assess the relative contributions of plasma and mitochondrial membrane potentials (Ψp and Ψm, respectively) to overall fluorescence. Addition of synaptosomes to media containing 0.5 μM dye caused a decrease in fluorescence intensity due to dye accumulation, which equilibrated usually within 5 min. Depolarization of mitochondria by combined treatment with cyanide and oligomycin increased fluorescence by 42%, indicating significant prior accumulation of dye into intrasynaptosomal mitochondria. Ψp was calculated to be –54 mV and was not altered significantly by prior depolarization of Ψm with cyanide and oligomycin (hereafter referred to as “poisoned”synaptosomes). Similarly, the linear relationship between dye fluorescence and Ψp was not altered by depolarization of Ψm. Valinomycin, a K+ ionophore, caused a Ψp-dependent increase in fluorescence in control (nonpoisoned) synaptosomes, but did not alter fluorescence of poisoned synaptosomes except when the extracellular concentration of K+ ([K+]e) was 2 mM, in which case valinomycin hyperpolarized Ψp by about 5 mV. The poreforming antibiotic gramicidin depolarized both Ψp and Ψm maximally. Under these conditions, Triton X-100 further increased fluorescence by 40%, indicating significant dye binding to synaptosomal components. In poisoned synaptosomes depolarized by 75 mM K+, gramicidin caused a decrease in fluorescence intensity (hyperpolarization of Ψp). The organic solvent dimethyl sulfoxide, used as a vehicle for the hydrophobic ionophores, had voltage-dependent effects on Ψp and Ψm. The magnitude of the fluorescence response of synaptosomes to depolarization with sufficient K+ to increase [K+]e by 40 mM was dependent on the initial Ψp and was not altered by prior depolarization of Ψm. Thus, K+-induced depolarization may be useful for estimating Ψp in the face of fluorescence change associated with depolarization of both Ψp and Ψm. Thus, carbocyanine dyes may be used to differentiate effects of experimental treatments on synaptosomal Ψp as compared with intrasynaptosomal Ψm.

Notes: Abstract The effects of castor oil and ricinoleic acid on small bowel electrical activity were studied in the fasted conscious dog and were compared to the effects elicited by two nonlaxative oils (triolein and oleic acid). Spike potential activity was monitored at two jejunal sites using unipolar recording electrodes. Castor oil, ricinoleic acid, and triolein produced an increased incidence of basic electrical rhythm (BER) with associated spike potentials when compared to a fasted control; however, the total electrical spiking activity produced by these oils was not statistically different from that induced by feeding. No treatment altered any of the characteristics of BER. A novel pattern of electrical spiking activity was observed in response to the laxatives. This pattern consisted of short repetitive bursts of spike potentials which migrated the length of the recording site. The laxative-induced electrical pattern persisted for several days after treatment with ricinoleic acid or castor oil, and interdigestive patterns were occasionally interrupted for as long as 72 hr. Electrical activity following feeding or the nonlaxative oils consisted of random spike potentials, and normal interdigestive electrical activity resumed within 24 hr. The laxative-induced electrical pattern was shown to be quantitatively distinct from those produced by feeding, fasting, or nonlaxative oils. This pattern may reflect an action of these laxatives on intestinal motility during a diarrheal state.