Effects of amphotericin B on the electrical properties ofNecturus gallbladder: Intracellular microelectrode studies

Summary Intracellular microelectrode techniques were employed to study the mechanism by which amphotericin B induces a transient mucosa-negative transepithelial potential (V _ms) in the gallbladder ofNecturus. When the tissue was incubated in standard Na-Ringer's solution, the antibiotic reduced the apical membrane potential by about 40 mV, and the basolateral membrane potential by about 35 mV whereas the transepithelial potential increased by about 5 mV. The electrical resistance of the apical membrane fell by 83%, and that of the basolateral membrane by 40%; the paracellular resistance remained unchanged. Circuit analysis indicated that the equivalent electromotive forces of the apical and basolateral membranes fell by 35 and 11 mV, respectively. Changes in potentials and resistances produced by ionic substitutions in the mucosal bathing medium showed that amphotericin B produces a nonselective increase in apical membrane small monovalent cation conductance (K, Na, Li). In the presence of Na-Ringer's on the mucosal side, this resulted in a reduction of the K permselectivity of the membrane, and thus in a fall of its equivalent emf. During short term exposure to amphotericin B,P _Na/P _Cl across the paracellular pathway did not change significantly, whereasP _K/P _Na doubled. These results indicate that V _ms is due to an increase of g_Na across the luminal membranes of the epithelial cells (Cremaschiet al., 1977,J. Membrane Biol. 34:55); the data do not support the alternative hypothesis (Rose & Nahrwold, 1976.J. Membrane Biol. 29:1) that V _ms results from a reduction in shuntP _Na/P _Cl acting in combination with a rheogenic basolateral Na pump.     

Light-dependent conformational change at rhodopsin's cytoplasmic surface detected by increased susceptibility to proteolysis

Rhodopsin in bovine photoreceptor disk membranes was subjected to limited proteolysis by thermolysin, removing twelve amino acids from rhodopsin's carboxyl terminus. (1) The rate of proteolysis is significantly faster with rhodopsin following exposure to light than with unbleached rhodopsin, provided that the incubation conditions (pH, temperature) favor the formation of metarhodopsin II. (2) If the disk membranes are illuminated under conditions in which metarhodopsin I is the predominant photoproduct (pH 8.5, 0°C), no increase in the rate of proteolysis is observed compared to unilluminated membranes. (3) The light-induced increase in the rate of proteolysis is transient: it slowly decays in the dark to the original rate found for unbleached rhodopsin. The enhanced susceptibility to proteolysis appears to measure a conformational change at rhodopsin's cytoplasmic surface which is first exhibited at the metarhodopsin II stage. This and possibly other light-dependent changes may allow rhodopsin to mediate its signal as a light-receptor protein by binding to and activating certain rod cell enzymes.     

A collagen-like glycoprotein from elastin-rich tissues

Extracts of bovine aorta and nuchal ligament contain several large glycoproteins. The major glycoprotein species has been isolated and has been shown to be collagenase sensitive with an apparent molecular weight of 140,000 daltons. The protein exists in disulphide-bonded aggregates, contains hydroxyproline and hydroxylysine in 1:1 ratio and is unlike any of the known collagen types in amino acid analysis. Its presencein ligament extracts indicates that it is not derived from basement membranes. The evidence suggests that this protein is not derived from the microfibrillar components of the elastic tissues.     

Aldosterone regulation of active sodium chloride transport in the lizard colon (Gallotia galloti)

Summary Bioelectrical parameters and unidirectional sodium and chloride fluxes were measured under voltageclamp conditions in groups of lizards submitted to single or chronic aldosterone treatment. Both acute (AT) and chronic (CT) treatment induced significant increases in the short-circuit current (I _sc), as well as in the mucosa-to-serosa (J _m-s ^Na ) and net sodium flux (J _net ^Na ). In AT tissues, aldosterone did not change net chloride flux (J _net ^Cl ) but did so in CT tissues. Amiloride reduced the aldosterone-increased I _sc in AT and CT tissues, inhibited J _net ^Na in AT tissues and abolished it in CT colons. J _net ^Cl was also reduced by the diuretic in the group of AT colons, whereas no changes were observed in the CT tissues. Addition of luminal DIDS reduced Na⁺ absorption and totally inhibited Cl^- absorption in the AT tissues, but did not change I _sc. However, in CT tissues neither Na⁺ nor Cl^- transport were affected by DIDS. A good relationship between I _sc and J _m-s ^Na was apparent after DIDS treatment in AT tissues. In this group, simultaneous addition of DIDS and amiloride totally abolished J _net ^Na and reduced I _sc to untreated control values. Addition of serosal ouabain abolished I _sc and Na⁺ absorption in AT and CT colons, but Cl^- absorption was only altered in AT tissues. These results support the hypothesis that aldosterone induces an electrogenic, amiloride-sensitive sodium absorption, and in a dose-dependent fashion suppresses electroneutral NaCl absorption in the lizard colon.Abbreviations AT acutely treated - CT chronically treated animals - DIDS 4-4-diisothiocyanatostibene-2-2-disulfonic acid - DMSO dimethylsulphoxide - G _t tissue conductance - I _sc short circuit current - PD transepithelial potential difference - SITS 4-acetamido-4-isothiocyanatostilbene-2-2-disulfonic acid - UC untreated controls Preliminary results of this paper were presented at the X ^th meeting of the European Intestinal Transport Group (EITG), Askov Hojskole, Denmark, 16–19 September 1990     

On the origin of mitochondria: a reexamination of the molecular structure and kinetic properties of pyruvate dehydrogenase complex from brewer''s yeast

45Ca2+ incorporated in response to glucose was selectively mobilized from the beta-cell-rich pancreatic islets of ob/ob-mice after raising the intracellular Na+ by removal of K+ or addition of ouabain or veratridine. Also studies of insulin release indicated opposite effects of glucose and Na+ on the intracellular sequestration of calcium. The fact that glucose inhibits insulin release induced by raised intracellular Na+ indicates that this sugar can lower the cytoplasmic [Ca2+]. The concept of a dual action of glucose on the cytoplasmic [Ca2+]. The concept of a dual action of glucose on the cytoplasmic [Ca2+] might well explain previous observations of an inhibitory component in the glucose action on the 45Ca2+ efflux.     

Solubilization and partial purification of protein kinase systems from brain membranes that phosphorylate calspectin: A spectrin-like calmodulin-binding protein (fodrin)

In brain tissue a spectrin-like calmodulin-binding protein calspectin, or fodrin, is concentrated in a synaptosome fraction, where most of the calspectin is associated with the synaptic membranes. This endogenous calspectin was phosphorylated by protein kinase system(s) associated with the membranes. Here, we report the solubilization and partial purification of the membrane-associated calspectin kinase activity. The activity was resolved on a gel filtration column into two fractions, peaks I and II having estimated M_r of 800 000 and 88 000. The activity of peak I was dependent on the presence of both Ca²⁺ and calmodulin. Peak II revealed a basal activity in the absence of Ca²⁺ and calmodulin, which was stimulated 2-fold by addition of Ca²⁺. Calmodulin had no effect on the peak II activity.     

The antimutagenic effect of selenium on 7,12-dimethylbenz(a)anthracene and metabolites in the amesSalmonella/microsome system

The antimutagenic effect of selenium as sodium selenite, sodium selenate, selenium dioxide, and seleno-methionine was studied in the AmesSalmonella/microsome mutagenicity test using 7,12-dimethylbenz(a)anthracene (DMBA) and some of its metabolites. Selenium (20 ppm) as sodium selenite reduced the number of histidine revertants on plates containing up to 100 μg DMBA/plate. Increasing concentrations of selenium as sodium selenite, sodium selenate, and selenium dioxide up to 40 ppm Se progressively decreased the number of revertants caused by 50 μg DMBA. DMBA and its metabolites 7-hydroxymethyl-12-methylbenz(a)anthracene, 12-hydroxymethyl-7-methylbenz(a)anthracene, and 3-hydroxy-7,12-dimethylbenz(a)anthracene were mutagenic forSalmonella typhimurium TA100 in the presence of an S-9 mixture. Selenium supplementation as Na₂SeO₃ reduced the number of revertants induced by these metabolites to background levels. The antimutagenic effect of inorganic selenium compounds cannot be explained by toxicity of selenium as determined by viability tests withSalmonella typhimurium TA100. Selenium supplementation in all forms examined, except sodium selenate, decreased the rate of spontaneous reversion. Selenium as sodium selenate was slightly mutagenic at concentrations of 4 ppm or less. Higher concentration of Na₂SeO₄ inhibited the mutagenicity of DMBA. The present studies support the anticarcinogenic potential of selenium and indicate that form and concentration are important factors in this trace element's efficacy.     

Yeast mitochondrial and cytoplasmic valyl-tRNA synthetases

Yeast mitochondrial tRNA synthetase has been partially purified and chromatographic, catalytic and antigenic properties have been compared to the cytoplasmic homologous enzyme from yeast. No significant differences could be observed between the two enzymes with respect to their behaviour during ammonium sulfate precipitation or in chromatographic separation on DEAE cellulose, hydroxylapatite and Sephadex G 200. The Km of the two enzymes toward tRNAs from yeast mitochondria, yeast cytoplasm or E. coli are pratically identical. The antigenic properties of the two enzymes are very similar; antisera against either the mitochondria or the cytoplasmic enzyme lead to the inhibition of their catalytic properties. The mitochondrial ValRS is formed by a single polypeptide chain whose molecular weight is 125,000 daltons, a value very close to that of the yeast cytoplasmic enzyme.