Inactivation kinetics and pharmacology distinguish two calcium currents in mouse pancreatic B-cells

Summary Voltage-dependent calcium currents were studied in cultured adult mouse pancreatic B-cells using the whole-cell voltage-clamp technique. When calcium currents were elicited with 10-sec depolarizing command pulses, the time course of inactivation was well fit by the sum of two exponentials. The more rapidlyinactivating component had a time constant of 75±5 msec at 0 mV and displayed both calcium influx- and voltage-dependent inactivation, while the more slowly-inanctivating component had a time constant of 2750±280 msec at 0 mV and inactivated primarily via voltage. The fast component was subject to greater steady-state inactivation at holding potentials between –100 and –40 mV and activated at a lower voltage threshold. This component was also significantly reduced by nimodipine (0.5 m) when a holding potential of –100 mV was used, whereas the slow component was unaffected. In contrast, the slow component was greatly increased by replacing external calcium with barium, while the fast component was unchanged. Cadmium (1–10 m) displayed a voltage-dependent block of calcium currents consistent with a greater effect on the high-threshold, more-slowly inactivating component. Taken together, the data suggest that cultured mouse B-cells, as with other insulin-secreting cells we have studied, possess at least two distinct calcium currents. The physiological significance of two calcium currents having distinct kinetic and steady-state inactivation characteristics for B-cell burst firing and insulin secretion is discussed.     

Distribution of Na+, K+-ATPase α-Subunit Isoforms in Rat Pituitary

The distributions of alpha-subunit isoforms of the Na+,K(+)-ATPase in rat pituitary were determined by immunoblotting and immunohistochemistry. Immunoreactivity for all three forms is present in the neural lobe, whereas the anterior lobe contains only alpha 1 and alpha 2. Most areas of the intermediate lobe exhibit faint immunoreactivity for only alpha 1, but thin strands of cells which stain strongly for all three isoforms are also present in this lobe. The previously reported ouabain inhibitable Na+,K(+)-ATPase activity in the neural lobe is consistent with the presence of both alpha 2 and alpha 3 subunits.     

Expression of Transferrin mRNA in the CNS of Normal and Jimpy Mice

Both the iron mobilization protein transferrin and iron itself are found predominantly in oligodendrocytes in the brain and consequently have been hypothesized to have a role in myelination. This study is designed to begin to understand the mechanism(s) that control the expression of transferrin at the gene level in the nervous system using a hypomyelinating murine mutant (jimpy mouse). With this animal model it is possible to determine if transferrin gene expression in the nervous system is dependent on the presence of a mature oligodendrocytic population. The results demonstrate that normally expression of the transferrin gene increases from postnatal day 5 to 22-25 and then levels off in the adult. In the jimpy mouse, the relative amount of transferrin gene expression is less than that of littermate controls at 5 days of age. Furthermore, transferrin gene expression does not increase with age beyond the level observed at postnatal day 5 in the jimpy mouse. It is concluded from this study that the majority of the transferrin mRNA in the mouse brain is expressed by and/or requires the presence of a mature oligodendrocytic population.     

A remnant New Zealand carr

Carr vegetation was once extensive in New Zealand. It can be divided into Cordyline australis I Carex secta carr, comprising an open wood with scattered large herbs and an abundance of Carex species, and podocarp carr, dominated by tall conifers. Both have been almost eliminated by agricultural development. We studied a remnant of Cordyline australis I Carex secta carr in South Island, which graded at one end into salt marsh. Eight communities were recognized, including one pure salt marsh and two more with saline influence. Near the stream that provided moisture and alluvium for the area were herbaceous communities. In the poorly-drained area away from the stream were communities dominated by shrubs and small trees. The soil environment is similar to that of many European carrs, though generally less organic. A number of the exotic species are also found in European carrs.     

Pre-column derivatization of free bile acids for high-performance liquid chromatographic and gas chromatographic—mass spectrometric analysis

Pentachlorophenyl (PCP) esters of five free bile acids (FBA) were obtained by reacting the FBA and Kovacs' complex (KC) in a 1:8 molar ratio in acetone at 65°C, and were purified by column chromatography on silica gel. The esters were crystallized from benzene—hexane, derivatized as trimethylsilyl ethers for gas chromatography on a DB-1 capillary column and for gas chromatography—mass spectrometry with a DB-5 column, and mass spectrometry (MS) in the electron-impact (EI) positive-ion mode at 70 eV. The reaction is specific for FBA even in the presence of glycine and taurine conjugates of bile acids. The PCP esters were treated with benzylamine in chloroform or methanol to produce N-benzyl derivatives of FBA. The N-benzylamides were separated by high-performance liquid chromatography (HPLC) on a 4-μm Nova-Pak C₁₈ column, studied by thermospray—LC—MS, and in the direct insertion probe—EI positive-ion mode.     

Conformational changes of recombinant human granulocyte-colony stimulating factor induced bypH and guanidine hydrochloride

Fluorescence and circular dichroism were used to follow thepH-dependent conformational changes of granulocyte colony stimulating factor (G-CSF). Tryptophan fluorescence of the spectra monitored at 344 nm, or after deconvolution of the emission spectra, at 345 nm, showed a decrease in intensity on going frompH 7 to 4, with a midtransitionpH of 5.8. On the other hand, tyrosine fluorescence measured either by the ratio of intensity at 308 nm to that at 344 nm, or by the fluorescence intensity at 303 nm after deconvolution of the spectra, increased in intensity as thepH was changed from 6 to 2.5, with a midtransitionpH of 4.5. Near UV circular dichroic spectra also showed changes betweenpH 7.5 and 4.5, which correlated with the transition monitored by the tryptophan fluorescence. The guanidine hydrochloride-induced conformational changes of G-CSF at fivepH values from 2.5 to 7.5 were also studied. Circular dichroic and fluorescence spectra revealed minor conformational changes by the addition of 1 or 2 M guanidine HCl at allpH values examined, while the major conformational transition occurred between 2 and 4 M guanidine hydrochloride. The secondary structure of the protein was most stable betweenpH 3.3 and 4.5. The guanidine HCl-induced denaturation of G-CSF involved more than a two-state transition, with detectable intermediate(s) present, and the structure of the intermediate(s) appeared to depend on thepH used. These results are consistent with thepH dependence of the structure described above, and demonstrate the complex conformational properties of G-CSF.     

Stimulation of bacterial cytokinesis by bacteriophage predation

Bacteriophage (phage) are ubiquitous in the water column and in the sediments of most natural water. Because of their colloidal nature, they can either aggregate into clumps large enough to settle into the sediment or departing upon the physiochemical conditions or disassociate and reenter the water column. About 80% of the bacterial strains isolated from New River sediment have a virulent phage that can be isolated with them.Liquid cultures of a strain of Pseudomonas aeruginosa isolated from the New River along with its phage were set up. One was infected with the virulent phage and another kept as a control. Daily counts were made of bacterial numbers. After 10 days the control culture was infected and counted for 3 more days.Both cultures divided exponentially at first. The infected culture continued to divide at about half the initial rate. The uninfected culture nearly ceased division, but when phage were added it quickly began to divide.The virulent phage infection clearly stimulated host division. The effect was to establish itself as an endemic infection which did not outpace its host's division rate. Further, the enhanced division rate may act to increase the host's share of available nutrients and benefit its competitive position in the system.     

Deficiency in integrin-mediated transmembrane signaling and microfilament stress fiber formation by aging dermal fibroblasts from normal and down''s syndrome patients

Previous evidence has shown a deficiency in microfilament stress fiber formation upon short-term cycloheximide treatment of cultured human dermal fibroblasts while cytoplasmic spreading appeared completely normal and other cytoskeletal networks organized normally. This deficiency applied to collagen substrata (not fibronectin substrata) and was specific for in vitro-aged normal fibroblasts and for fibroblasts from three different Down's syndrome patients at any passage level. To identify the mechanism(s) for matrix receptor deficiency in aging cells, cells were evaluated for amounts and distributions of several integrin subunits using specific monoclonal antibodies and two complementary experimental approaches. Flow cytometric analyses have shown that all these cells at all passage levels have large amounts of alpha 3 and beta 1 integrin subunits and smaller amounts of the alpha 5 subunit, directed to fibronectin, which are minimally affected in their cell surface availability by cycloheximide treatment. In contrast, cycloheximide treatment leads to the loss from surface availability of most of the alpha 2 subunit, directed to collagen, in late-passage papillary and reticular normal fibroblasts and in all three Down's patient cells at all passages. Prior growth of cells in ascorbate-supplemented medium, which overcomes the deficiency in stress fiber formation, conserves the large amounts of cell surface-available alpha 2 subunit detectable by flow cytometry. When amounts of integrin subunits were evaluated by immunoprecipitation of [35S]methionine-radiolabeled cells, there was no diminution of the alpha 2 subunit or any other subunit for any cells upon cycloheximide treatment; however, there was much less alpha 2 subunit complexed with beta 1 in aging normal and Down's cells. Therefore, cycloheximide treatment does not lead to loss in the amounts of the alpha 2 subunit but rather to its masking at the cell surface and inability to transmit signals across the plasma membrane to effect stress fiber formation. This aging-related deficiency in integrin-mediated signaling can now be studied mechanistically with a variety of approaches to determine the nature of cell-surface molecules interacting with integrins (cis- and/or trans-acting molecules) that discriminate functional from nonfunctional receptors.     

Evidence that the Loss of the Voltage-Dependent Mg2+ Block at the N-Methyl-D-Aspartate Receptor Underlies Receptor Activation During Inhibition of Neuronal Metabolism

Both phosphointermediate- and vacuolar-type (P- and V-type, respectively) ATPase activities found in cholinergic synaptic vesicles isolated from electric organ are immunoprecipitated by a monoclonal antibody to the SV2 epitope characteristic of synaptic vesicles. The two activities can be distinguished by assay in the absence and presence of vanadate, an inhibitor of the P-type ATPase. Each ATPase has two overlapping activity maxima between pH 5.5 and 9.5 and is inhibited by fluoride and fluorescein isothiocyanate. The P-type ATPase hydrolyzes ATP and dATP best among common nucleotides, and activity is supported well by Mg2+, Mn2+, or Co2+ but not by Ca2+, Cd2+, or Zn2+. It is stimulated by hyposmotic lysis, detergent solubilization, and some mitochondrial uncouplers. Kinetic analysis revealed two Michaelis constants for MgATP of 28 microM and 3.1 mM, and the native enzyme is proposed to be a dimer of 110-kDa subunits. The V-type ATPase hydrolyzes all common nucleoside triphosphates, and Mg2+, Ca2+, Cd2+, Mn2+, and Zn2+ all support activity effectively. Active transport of acetylcholine (ACh) also is supported by various nucleoside triphosphates in the presence of Ca2+ or Mg2+, and the Km for MgATP is 170 microM. The V-type ATPase is stimulated by mitochondrial uncouplers, but only at concentrations significantly above those required to inhibit ACh active uptake. Kinetic analysis of the V-type ATPase revealed two Michaelis constants for MgATP of approximately 26 microM and 2.0 mM. The V-type ATPase and ACh active transport were inhibited by 84 and 160 pmol of bafilomycin A1/mg of vesicle protein, respectively, from which it is estimated that only one or two V-type ATPase proton pumps are present per synaptic vesicle. The presence of presumably contaminating Na+,K(+)-ATPase in the synaptic vesicle preparation is demonstrated.     

In Vivo Evidence that Lithium Inactivates Gi Modulation of Adenylate Cyclase in Brain

In vivo microdialysis of cyclic AMP from prefrontal cortex complemented by ex vivo measures was used to investigate the possibility that lithium produces functional changes in G proteins that could account for its effects on adenylate cyclase activity. Four weeks of lithium administration (serum lithium concentration of 0.85 +/- 0.05 mM; n = 11) significantly increased the basal cyclic AMP content in dialysate from prefrontal cortex of anesthetized rats. Forskolin infused through the probe increased dialysate cyclic AMP, but the magnitude of this increase was unaffected by chronic lithium administration. Inactivation of the inhibitory guanine nucleotide binding protein Gi with pertussis toxin increased dialysate cyclic AMP in control rats, as did stimulation with cholera toxin (which activates the stimulatory guanine nucleotide binding protein Gs). The effect of pertussis toxin was abolished following chronic lithium, whereas the increase in cyclic AMP after cholera toxin was enhanced. In vitro pertussis toxin-catalyzed ADP ribosylation of alpha i (and alpha o) was increased by 20% in prefrontal cortex from lithium-treated rats, but the alpha i and alpha s contents (as determined by immunoblot) as well as the cholera toxin-catalyzed ADP ribosylation of alpha s were unchanged. Taken together, these results suggest that chronic lithium administration may interfere with the dissociation of Gi into its active components and thereby remove a tonic inhibitory influence on adenylate cyclase, with resultant enhanced basal and cholera toxin-stimulated adenylate cyclase activity.