Restoration Of Ca2 Research Articles

Occluding junctions and cytoskeletal components in a cultured transporting epithelium.

MDCK cells form uninterrupted monolayers and make occluding junctions similar to those of natural epithelia. This aricle explores the relationship between these junctions and the cytoskeleton by combining studies on the distribution of microfilaments and microtubules with the effect of drugs, such as colchicines and cytochalasin B, on the degree of tightness of the occluding junctions. To study the degree of tightness, monolayers were prepared by plating MDCK cells on mylon disks coated with collagen. Disks were mounted as flat sheets between two Lucite chambers, and the sealing capacity of the junctions was evaluated by measuring the electrical resistance across the monolayers. Equivalent monolayers on coverslips were used to study the distribution of microtubules and microfilaments by indirect immunofluorescence staining with antibodies against tubulin and actin. This was done both on complete cells and on cytoskeleton preparations in which the cell membranes had been solubilized before fixation. Staining with antiactin shows a reticular pattern of very fine filaments that spread radially toward the periphery where they form a continuous cortical ring underlying the plasma membrane. Staining with antitubulin depicts fibers that extend radially to form a network that occupies the cytoplasm up to the edges of the cell. Colchicine causes a profound disruption of microtubules but only a 27 percent decrease in the electrical resistance of the resting monolayers. Cytochalasin B, when present for prolonged periods, disrupts the cytoplasmic microfilaments and abolishes the electrical resistance. The cortical ring of filaments remains in place but appears fragmented with time. We find that removal of extracellular Ca(++), which causes the tight junctions to open, also causes the microfilaments and microtubules to retract toward the center of the cells. The process of junction opening and fiber retraction is reversed by the restoration of Ca(++). Colchicine has no effect on either the opening or reversal processes, but cytochalasin B inhibits the resealing of the junctions by disorganizing the filaments in the ring and at the apical border of the cells. These cytochalasin B effects are fully reversible. The correlation among cell shape, cytoskeletal patterns, and electrical resistance in the EGTA-opened and resealed monolayers suggests that microfilaments, through their association with plasma membrane components, play a role in positioning the junctional strands and influence the degree of sealing of the occluding junctions.

Isolation of anticoagulant proteins from cobra venom (Naja nigricollis). Identity with phospholipases A2.

Three anticoagulant proteins were isolated from the venom of Naja nigricollis (spitting cobra). The peaks of anticoagulant activity co-chromatographed with phospholipase A2 activities. The three proteins were homogeneous by the criteria of electrophoresis in two acidic polyacrylamide gel systems. Electrophoresis in sodium dodecyl sulfate also yielded single protein bands with molecular weights of about 15,000. The amino acid compositions of the three anticoagulant proteins are reported. All three proteins have only one amino acid replacement in the first 25 to 30 amino acids of their NH2-terminal sequences, compared to the sequence of the basic phospholipase from N. nigricollis venom. Removal of Ca2+ from the crude venom caused loss of both anticoagulant and phospholipase activities, and restoration of Ca2+ caused partial recovery of both activities. Both activities were lost in parallel when the venom was heated between pH 6.0 and 8.5. The association of the anticoagulant effect with phospholipase activity contradicts the previous conclusion that phospholipase is not responsible for the anticoagulant action of cobra venom. The previous results might be explained by independence of the anticoagulant and phospholipase effects within the same protein molecule, or by different activity levels of monomer and dimer forms of the enzyme.

Superficial cell-wall layers on Spirillum "Ordal" and their in vitro reassembly.

The cell envelope of Sporillum sp. strain "Ordal" (possibly a variety of S. anulus) demonstrated multiple superficial wall layers which were diverse in their macromolecular arrays. Negative staining and freeze-etching techniques revealed an outer hexagonally packed layer and an inner tetragonally packed layer. However, both thin sections and freeze-etched cleavages of the wall showed that each of these regular structures rested upon a backing layer, and that there was a delicate amorphous layer overlying the outer hexagonal array. Rotary integration, optical deffraction, and reconstruction of image were used to clarify measurements of each array and to verify the validity of a diagrammatic model of the outer hexagonal system. The integrity of these layers required suitable cations (Ca2+ appeared essential) and pH (pH less than or equal to 4.6 dissociated most superficial layers). These observations aided in the development of a low-pH cationic-substitution technique, in which Na+ replaced essential Ca2+, for extraction of the layers from the cell surface. Dialysis to remove Na+ and restoration of Ca2+ initiated in vitro reassembly of the superficial layer components until regularly structured assembly products were formed.

Calcium-secretagogue interaction in the stimulation of gastric acid secretion.

SummaryGastric epithelia were depleted of Ca2+ in the continued presence of secre-tagogues by removing this cation from the bathing media and repeated washouts with or without EDTA until acid secretion was reduced to zero. Under these conditions, both the transmucosal PD and resistance declined. Incubation of these epithelia in presence of 1 mM Ca2+ in the luminal bathing medium resulted in a partial reversal of PD and resistance with initiation of some secretion, in contrast to near complete reversal obtained following restoration of Ca2+ to the serosal bathing medium. Dibutyryl cyclic-AMP or theophylline enhance acid secretion in these circumstances without complete reversal of PD and resistance. These observations suggest that cellular Ca2+ may play a direct role in the production of H+per se by mediating the secretagogue effect in raising intracellular cyclic-AMP levels, in addition to its established role in the maintenance of integrity of junctional complexes which are essential for the luminal segr...

Effect of Phospholipases on the Structure and Function of Mitochondria

Abstract 1. Exposure of bovine heart mitochondria to purified preparations of phospholipase A or phospholipase C resulted in losses of phosphorylation capacity, whereas respiration was not impaired. 2. The inhibitory effects of low concentrations of phospholipase A on phosphorylation were prevented or reversed by bovine serum albumin. At higher concentrations of phospholipase A neither bovine serum albumin nor lecithin were effective in restoring phosphorylation. 3. Exposure of mitochondria to phospholipase C resulted in a loss of Ca++ translocation, a partial depression of oxidative phosphorylation, and a partial loss of organically bound phosphorus. 4. Phospholipase C-treated mitochondria were resistant to the uncoupling action of Ca++ or K+ plus valinomycin. Addition of nigericin together with valinomycin and K+ was required to abolish phosphorylation associated with oxidation in phospholipase C-treated mitochondria. These responses resemble those of submitochondrial particles rather than those of mitochondria. In electron micrographs, phospholipase C-treated mitochondria looked like submitochondrial particles packaged in mitochondria. 5. A partial restoration of Ca++ translocation after phospholipase C digestion was achieved by addition of either egg lecithin or of large amounts of bovine serum albumin.

Cytochrome oxidase solubilization at high pH: I. Effects of pH, Ca 2+ and Mg 2+ on the reversible solubilization of cytochrome oxidase and cytochrome b by synthetic zeolites and by EDTA

Treatment of extensively water-washed beef heart or yeast mitochondria with synthetic A or X type zeolites in the Na + or K + form disaggregates the mitochondria and solubilizes cytochromes b, c, and oxidase in aqueous systems whose pH is between 10 and 11. However, when mitochondria are treated with the same types of A or X zeolites in the Ca 2+ or Mg 2+ forms, only solubilization of some cytochrome c occurs, and cytochromes b and oxidase remain insoluble in aqueous systems whose pH is between 8 and 9.5. Incubation of heart mitochondria with 0.05 m EDTA at pH 10.5 (or yeast mitochondria at pH values between 9 and 10) results in complexation of all of the mitochondrial Ca 2+ and Mg 2+ by the EDTA, and concomitantly, cytochromes b, c, and oxidase are solubilized. At lower pH values, the divalent cations are still complexed, but solubilization of oxidase and b components does not occur. Restoration of Ca 2+ (as chloride) to either zeolite- or EDTA-solubilized mitochondrial systems quantitatively precipitates the b, c, and oxidase components which can be centrifuged down at 2000 g for 10 min. However, restoration of Mg 2+ (as chloride) to either zeolite- or EDTA-solubilized mitochondrial systems causes slow formation of an aggregate which does not contain cytochrome b, c, or oxidase components, all of which remain in solution. Finally, when the high pH of zeolite- or EDTA-solubilized cytochrome b and oxidase systems is lowered by back-titration with acid, the above components are quantitatively precipitated.