Basement membrane collagen requirements for attachment and growth of mammary epithelium.

In vivo mammary epithelial cells rest upon a basement membrane composed in part of type IV collagen which is synthesized by these cells. In this study, basement membrane collagen is shown to be selectively recognized by normal mammary ducts and alveoli for attachment and growth when compared to the types of collagen derived from stroma (types I or III) or cartilage (type II). Cell attachment and growth on type I collagen is inhibited by the proline analogue, cis-hydroxyproline, which blocks normal collagen production. These effects of cis-hydroxyproline are not apparent when a basement membrane collagen substratum is provided. Unlike normal mammary epithelium, mammary fibroblasts show little preference for the collagen to which they will attach. A requirement of type IV collagen synthesis for normal mammary epithelial cell attachment and growth on stromal collagen in vitro may have significance in vivo where a basement membrane scaffold may be necessary for normal mammary morphogenesis and growth.     

The Surface Charge of Isolated Toad Bladder Epithelial Cells : Mobility, effect of pH and divalent ions

The surface charge of epithelial cells isolated from the toad bladder has been determined by the microscope method of cell electrophoresis. The cells possess a net negative charge, and a net surface charge density of 3.6 x 10⁴ electronic charges per square micron at pH 7.3. Estimates of net surface charge over the alkaline pH range indicate (a) that an average distance of the order of 40 A separates the negatively charged groups, and (b) that amino as well as acid groups are present at the electrophoretic surface of shear. A significant increase in mobility following cyanate treatment of the cells suggests that a large proportion of the amino groups are the ε-amino groups of lysine. In view of the known effects of calcium and other divalent ions on cell permeability and cell adhesion, the extent of binding of calcium and magnesium to the cell surface was determined by the electrophoretic technique. Mobility was significantly decreased in the presence of calcium or magnesium, indicating that these ions are bound by surface groups. When the pH was lowered from 7.3 to 5.2, calcium binding was markedly decreased, an observation consistent with competition between calcium and hydrogen ions for a common receptor site.     

Analysis of the effects of calcium or magnesium on voltage-clamp currents in perfused squid axons bathed in solutions of high potassium

Isolated axons from the squid, Dosidicus gigas, were internally perfused with potassium fluoride solutions. Membrane currents were measured following step changes of membrane potential in a voltage-clamp arrangement with external isosmotic solution changes in the order: potassium-free artificial seawater; potassium chloride; potassium chloride containing 10, 25, 40 or 50, mM calcium or magnesium; and potassium-free artificial seawater. The following results suggest that the currents measured under voltage clamp with potassium outside and inside can be separated into two components and that one of them, the predominant one, is carried through the potassium system. (a) Outward currents in isosmotic potassium were strongly and reversibly reduced by tetraethylammonium chloride. (b) Without calcium or magnesium a progressive increase in the nontime-dependent component of the currents (leakage) occurred. (c) The restoration of calcium or magnesium within 15–30 min decreases this leakage. (d) With 50 mM divalent ions the steady-state current-voltage curve was nonlinear with negative resistance as observed in intact axons in isosmotic potassium. (e) The time-dependent components of the membrane currents were not clearly affected by calcium or magnesium. These results show a strong dependence of the leakage currents on external calcium or magnesium concentration but provide no support for the involvement of calcium or magnesium in the kinetics of the potassium system.     

δ-Endotoxins induce cation channels in Spodoptera frugiperda brush border membranes in suspension and in planar lipid bilayers

Membrane potential measurements using a fluorescent dye indicated that two specific toxins active against Spodoptera frugiperda larvae (CryIC and CryID) cause immediate permeability changes in midgut epithelial brush border membrane vesicles (BBMV). The initial response and the sustained permeability change are cationic, notvery K⁺ selective, and occur at in vivo lethal doses (nM). The toxin response has a different ion selectivity and is more sensitive to Ba²⁺ than the intrinsic cation permeability of BBMV. Experiments incorporating BBMV into planar lipid bilayers (PLB) demonstrated that these vesicles contain cation channels (31, 47 and 76 pS). A 2–40 fold conductance increase was induced by nM concentrations of toxin in PLB containing BBMV. Cationic single channel transitions of 50, 106, 360 and 752 pS were resolved. Thus, Bacillus thuringiensis δ-endotoxins induce an increase in cation membrane permeability involving ion channels in BBMV-containing functional receptors.     

Regulation of intestinal epithelial intercellular adhesion and barrier function by desmosomal cadherin desmocollin-2

The role of desmosomal cadherin desmocollin-2 (Dsc2) in regulating barrier function in intestinal epithelial cells (IECs) is not well understood. Here, we report the consequences of silencing Dsc2 on IEC barrier function in vivo using mice with inducible intestinal–epithelial-specific Dsc2 knockdown (KD) (Dsc2^ERΔIEC). While the small intestinal gross architecture was maintained, loss of epithelial Dsc2 influenced desmosomal plaque structure, which was smaller in size and had increased intermembrane space between adjacent epithelial cells. Functional analysis revealed that loss of Dsc2 increased intestinal permeability in vivo, supporting a role for Dsc2 in the regulation of intestinal epithelial barrier function. These results were corroborated in model human IECs in which Dsc2 KD resulted in decreased cell–cell adhesion and impaired barrier function. It is noteworthy that Dsc2 KD cells exhibited delayed recruitment of desmoglein-2 (Dsg2) to the plasma membrane after calcium switch-induced intercellular junction reassembly, while E-cadherin accumulation was unaffected. Mechanistically, loss of Dsc2 increased desmoplakin (DP I/II) protein expression and promoted intermediate filament interaction with DP I/II and was associated with enhanced tension on desmosomes as measured by a Dsg2-tension sensor. In conclusion, we provide new insights on Dsc2 regulation of mechanical tension, adhesion, and barrier function in IECs.     

Fluorometric Assay of Potential Change of Bombyx mori Midgut Brush Border Membrane Induced by δ-Endotoxin from Bacillus thuringiensis

Bacillus thuringiensis δ-endotoxin, CryIA(a), increased ion permeability of brush border membrane vesicles isolated from midgut epithelia of Bombyx mori larvae. This ion permeability change was measured with a potential-sensitive fluorescent dye, 3,3′-dipropylthiadicarbocyanine iodide. This effect was observed at concentrations of the toxin that correspond to normal effective doses in vivo. CryIA(b) and heat-treat CryIA(a) did not show this effect. CryIA(a) did not show this effect on rat renal brush border membranes. The effects depended on the toxin dose in saturable manner. These suggest that this assay system reflects the mode of action of δ-endotoxin in vivo. The toxin increased various ion permeabilities, suggesting that δ-endotoxin forms non-selective cation pores on brush border membranes.     

AQP0-LTR of the Cat mouse alters water permeability and calcium regulation of wild type AQP0

Aquaporin 0 (AQP0) is the major intrinsic protein of the lens and its water permeability can be modulated by changes in pH and Ca²⁺. The Cataract Fraser (Cat^Fr) mouse accumulates an aberrant AQP0 (AQP0-LTR) in sub-cellular compartments resulting in a congenital cataract. We investigated the interference of AQP0-LTR with normal function of AQP0 in three systems. First, we created a transgenic mouse expressing AQP0 and AQP0-LTR in the lens. Expression of AQP0 did not prevent the congenital cataract but improved the size and transparency of the lens. Second, we measured water permeability of AQP0 co-expressed with AQP0-LTR in Xenopus oocytes. A low expression level of AQP0-LTR decreased the water permeability of AQP0, and a high expression level eliminated its calcium regulation. Third, we studied trafficking of AQP0 and AQP0-LTR in transfected lens epithelial cells. At low expression level, AQP0-LTR migrated with AQP0 toward the cell membrane, but at high expression level, it accumulated in sub-cellular compartments. The deleterious effect of AQP0-LTR on lens development may be explained by lowering water permeability and abolishing calcium regulation of AQP0. This study provides the first evidence that calcium regulation of AQP0 water permeability may be crucial for maintaining normal lens homeostasis and development.     

Morphological changes in the zonula adhaerens during embryonic development of chick retinal pigment epithelial cells

Summary Retinal pigment epithelial cells from chicks at various stages of development were examined by transmission electron microscopy to determine how the adult form of the zonula adhaerens, composed of subunits termed zonula adhaerens complexes, is acquired. During early stages of development, between embryonic day 4 and embryonic day 7, the intermembrane discs of zonula adhaerens complexes appear to be formed from material already present between the junctional membranes of the zonulae adhaerentes. In contrast, the cytoplasmic plaque material of the zonulae adhaerentes is difficult to detect before hatching; it is seen as a dense band along the junctional membranes at hatching and as individual subunits in register with the intermembrane discs in adult retinal pigment epithelial cells. After embryonic day 16, when the zonulae adhaerentes increase dramatically in size, single zonula adhaerens complexes are also present basal to the zonulae adhaerentes along the lateral cell membrane. This suggests that, during later stages of development, the junctions grow in size and/or turn over by the addition of pre-assembled zonula adhaerens complexes.Abbreviations CMB Circumferential microfilament bundle - ZA Zonula adhaerens - ZAC Zonula adhaerens complex - RPE Retinal pigment epithelium     

The resting membrane potential of Drosophila melanogaster larval muscle depends strongly on external calcium concentration

The resting membrane potential (RMP) of most cells is not greatly influenced by the transmembrane calcium gradient because at rest, the membrane has very low permeability to calcium. We have observed, however, that the resting membrane potential of muscle cells in the larval bodywall of Drosophila melanogaster varies widely as the external calcium concentration is modified. The RMP depolarized as much as 21.8 mV/mM calcium at low concentrations, and on average, about 10 mV/mM across a range typical of neurophysiological investigations. The extent to which muscle RMP varies has important implications for the measurement of synaptic potentials as well. Two parameters of excitatory junctional potential (EJP) voltage were compared across a range of RMPs. EJP amplitude (ΔV) and peak voltage (maxima) change as a function of RMP; on average, a 10 mV change in RMP elicits a 4-5 mV change in EJP amplitude and peak voltage. The influence of the calcium gradient on resting and synaptic membrane potentials led us to investigate the endogenous ion concentrations of larval hemolymph. In addition to the major monovalent ions and calcium, we report the first voltammetric analysis of magnesium concentration in larval fruit fly hemolymph.     

Mitochondrial calcium function and dysfunction in the central nervous system

The ability of isolated brain mitochondria to accumulate, store and release calcium has been extensively characterized. Extrapolation to the intact neuron led to predictions that the in situ mitochondria would reversibly accumulate Ca²⁺ when the concentration of the cation in the vicinity of the mitochondria rose above the ‘set-point’ at which uptake and efflux were in balance, storing Ca²⁺ as a complex with phosphate, and slowly releasing the cation when plasma membrane ion pumps lowered the cytoplasmic free Ca²⁺. Excessive accumulation of the cation was predicted to lead to activation of the permeability transition, with catastrophic consequences for the neuron. Each of these predictions has been confirmed with intact neurons, and there is convincing evidence for the permeability transition in cellular Ca²⁺ overload associated with glutamate excitotoxicity and stroke, while the neurodegenerative disease in which possible defects in mitochondrial Ca²⁺ handling have been most intensively investigated is Huntington's Disease. In this brief review evidence that mitochondrial Ca²⁺ transport is relevant to neuronal survival in these conditions will be discussed.     

Changes in the Membrane Permeability of Frog's Sartorius Muscle Fibers in Ca-Free EDTA Solution

The changes in the membrane permeability to sodium, potassium, and chloride ions as well as the changes in the intracellular concentration of these ions were studied on frog sartorius muscles in Ca-free EDTA solution. It was found that the rate constants for potassium and chloride efflux became almost constant within 10 minutes in the absence of external calcium ions, that for potassium increasing to 1.5 to 2 times normal and that for chloride decreasing about one-half. The sodium influx in Ca-free EDTA solution, between 30 and 40 minutes, was about 4 times that in Ringer's solution. The intracellular sodium and potassium contents did not change appreciably but the intracellular chloride content had increased to about 4 times normal after 40 minutes. By applying the constant field theory to these results, it was concluded that (a) P_Cl did not change appreciably whereas P_K decreased to a level that, in the interval between 10 and 40 minutes, was about one-half normal, (b) P_Na increased until between 30 and 40 minutes it was about 8 times normal. The low value of the membrane potential between 30 and 40 minutes was explained in terms of the changes in the membrane permeability and the intracellular ion concentrations. The mechanism for membrane depolarization in this solution was briefly discussed.     

Synthesis and solid-state structure of magnesium and calcium imidazole complexes

Three octahedral complexes built from N-alkylsubstituted imidazoles and magnesium as well as calcium chloride are reported. The obtained solid-state structures differ significantly from each other, depending on the size of the metal ion and the substituent on the imidazole. A chloro-bridged structure is found in the case of tert-butylimidazole, while for the iso-propylimidazole the calcium ion is coordinated by six imidazoles. For the smaller magnesium cation, we also found a sixfold coordination, but here only three of the iso-propylimidazoles coordinate to the magnesium, the three other positions are taken by water molecules.     

Permeability and Electrical Properties of Thin Lipid Membranes

We present and discuss the permeability and electrical properties of thin lipid membranes, and the changes induced in these properties by several agents added to the aqueous phases after the membranes have formed. The unmodified membrane is virtually impermeable to ions and small "hydrophilic" solutes, but relatively permeable to water and "lipophilic" molecules. These properties are consistent with those predicted for a thin film of hydrocarbon through which matter is transported by dissolving in the membrane phase and then diffusing through it. The effect of cholesterol in reducing the water and "lipophilic" solute permeability is attributed to an increase of the "viscosity" of the hydrocarbon region, thus reducing the diffusion coefficient of molecules within this phase. The selective permeability of the membrane to iodide (I^-) in the presence of iodine (I₂) is attributed to the formation of polyiodides (perhaps I₅ ^-), which are presumed to be relatively soluble in the membrane because of their large size, and hence lower surface charge density. Thus, I₂ acts as a carrier for I^-. The effects of "excitability-inducing material" and the depsipeptides (particularly valinomycin) on ion permeability are reviewed. The effects of the polyene antibiotics (nystatin and amphotericin B) on ion permeability, discussed in greater detail, are the following: (a) membrane conductance increases with the 10th power of nystatin concentration; (b) the membrane is anion-selective but does not discriminate completely between anions and cations; (c) the membrane discriminates among anions on the basis of size; (d) membrane conductance decreases extraordinarily with increasing temperatures. Valinomycin and nystatin form independent conductance pathways in the same membrane, and, in the presence of both, the membrane can be reversibly shifted between a cation and anion permeable state by changes in temperature. It is suggested that nystatin produces pores in the membrane and valinomycin acts as a carrier.     

On the substrate specificity of the red cell calcium pump

ATP-dependent active calcium transport in inside-out human red cell membrane vesicles is stimulated by magnesium essentially parallel with an increase in MgATP concentration. At a constant, low (1 μM) calcium concentration, increasing ATP and magnesium increase the maximum calcium transport rate irrespective of the constant or decreasing concentrations of CaATP present. K_Ca for calcium pumping is practically unchanged at variable ATP and magnesium concentrations. Free magnesium above 1–2 mM inhibits active calcium transport, probably through a direct interaction with the transport enzyme. Based on the experimental findings reported we suggest that the true, physiological substrate of the red cell calcium pump is MgATP.     

Basolateral Mg2+/Na+ exchange regulates apical nonselective cation channel in sheep rumen epithelium via cytosolic Mg2+

High potassium diets lead to an inverse regulation of sodium and magnesium absorption in ruminants, suggesting some form of cross talk. Previous Ussing chamber experiments have demonstrated a divalent sensitive Na(+) conductance in the apical membrane of ruminal epithelium. Using patch-clamped ruminal epithelial cells, we could observe a divalent sensitive, nonselective cation conductance (NSCC) with K(+) permeability > Cs(+) permeability > Na(+) permeability. Conductance increased and rectification decreased when either Mg(2+) or both Ca(2+) and Mg(2+) were removed from the internal or external solution or both. The conductance could be blocked by Ba(2+), but not by tetraethylammonium (TEA). Subsequently, we studied this conductance measured as short-circuit current (I(sc)) in Ussing chambers. Forskolin, IBMX, and theophylline are known to block both I(sc) and Na transport across ruminal epithelium in the presence of divalent cations. When the NSCC was stimulated by removing mucosal calcium, an initial decrease in I(sc) was followed by a subsequent increase. The cAMP-mediated increase in I(sc) was reduced by low serosal Na(+) and serosal addition of imipramine or serosal amiloride and depended on the availability of mucosal magnesium. Luminal amiloride had no effect. Flux studies showed that low serosal Na(+) reduced (28)Mg fluxes from mucosal to serosal. The data suggest that cAMP stimulates basolateral Na(+)/Mg(2+) exchange, reducing cytosolic Mg. This increases sodium uptake through a magnesium-sensitive NSCC in the apical membrane. Likewise, the reduction in magnesium uptake that follows ingestion of high potassium fodder may facilitate sodium absorption, as observed in studies of ruminal osmoregulation. Possibly, grass tetany (hypomagnesemia) is a side effect of this useful mechanism.     

Improved methods for reducing calcium and magnesium concentrations in tissue culture medium: Application to studies of lymphoblast proliferation in vitro

Summary We have compared several methods for reducing calcium and magnesium concentrations in tissue culture medium, with the objective of producing selective deficiency effects on the growth of mouse (L5178Y) and human (P1R) lymphoblasts. In experiments in which calcium- and magnesium-“free” McCoy’s medium was supplemented with 15% horse or fetal calf serum, enough calcium and magnesium was provided by serum to support normal lymphoblast growth rate. Either dialysis or chelating-resin treatment of horse or fetal calf serum reduced calcium and magnesium contents approximately 100-fold. Use of dialyzed sera resulted in reduced growth rate, although in most cases the reduction in growth could be attributed to other effects of dialysis on serum, inasmuch as growth in those experiments was not restored to normal by the addition of calcium and magnesium to the medium. In contrast, the reduction of lymphoblast growth rate that occurred when resin-treated serum was used was always attributable to removal of calcium and magnesium, as normal growth always occurred in cultures to which calcium and magnesium were added. To demonstrate a growth-inhibiting effect on either mouse or human lymphoblasts by severe reduction of either calcium or magnesium in the presence of normal amounts of the alternative cation, it was necessary to (a) expose McCoy’s Ca−Mg-“free” medium to chelating-resin to reduce further the residual cation concentrations; (b) wash cells from stock cultures in a medium devoid of calcium and magnesium prior to inoculation into experimental cultures; (c) reduce the proportion of serum in the final medium from 15 to 5%; and (d) add 100 μM EGTA to cultures. Under these conditions, growth of both cell types was completely abolished in the presence of normal magnesium but in the absence of added calcium, and markedly reduced in the presence of normal calcium but in the absence of magnesium. These modifications did not compromise growth in cultures containing normal concentrations of both ions. This work has been supported by a Research Grant from the U.S. Public Health Service (CA 12790), from the Monroe County Cancer and Leukemia Association, and by a contract from the Atomic Energy Project at the University of Rochester, and has been assigned publication no. UR-3490-624.     

Al and Ca Alteration of Membrane Permeability of Quercus rubra Root Cortex Cells

This study was undertaken to quantify the effect of aluminum and calcium on membrane permeability. The influence of Ca²⁺ (0.2-3.7 millimolar) and Al³⁺ (0-3.7 millimolar) on the permeability of root cortical cells of Quercus rubra was measured using three nonelectrolytes (urea, methyl urea, and ethyl urea) as permeators of progressively increasing lipid solubility. Water permeability was also measured. Al³⁺ (a) increased membrane permeability to the nonelectrolytes, (b) decreased the membrane's partiality for lipid permeators, and (c) decreased membrane permeability to water. Ca²⁺ had effects on permeability that were opposite to those of Al³⁺. When Al³⁺ and Ca²⁺ were tested in combination, these opposite effects counteracted each other. The results suggest that Al³⁺ altered the architecture of membrane lipids.     

Some Kinetic and Metabolic Characteristics of Calcium-Induced Potassium Transport in Human Red Cells

When fresh human erythrocytes or their ghosts are incubated with Ca + IAA (iodoacetic acid) + adenosine, K permeability increases; K permeability also increases when energy-depleted cells or their ghosts are incubated with Ca alone. Na transport decreases or remains unaltered in both situations. The Ca-induced increase in K permeability in the depleted cell system is qualitatively similar to that seen in the fresh cell system and furnishes a means for studying the metabolic dependence of calcium's action. Studies with the depleted system suggest that the normal refractiveness of the cell to calcium is provided by a metabolically dependent substrate. Removal of this substrate allows Ca to enter the cell and exert its effect. By using ⁴⁷Ca, a maximum value was obtained (3–7 x 10^-6 moles/liter of red blood cells) for the quantity of calcium that is taken up by the cell and responsible for the change in K permeability. Measurements of the unidirectional fluxes of K, obtained during the time Ca increases K permeability, appear to satisfy the flux ratio equation for passive diffusion through a membrane.     

The Degree of Resistance of Erythrocyte Membrane Cytoskeletal Proteins to Supra-Physiologic Concentrations of Calcium: An In Vitro Study

Calcium is a key regulator of cell dynamics. Dysregulation of its cytosolic concentration is implicated in the pathophysiology of several diseases. This study aimed to assess the effects of calcium on the network of membrane cytoskeletal proteins. Erythrocyte membranes were obtained from eight healthy donors and incubated with 250 µM and 1.25 mM calcium solutions. Membrane cytoskeletal proteins were quantified using SDS-PAGE at baseline and after 3 and 5 days of incubation. Supra-physiologic concentrations of calcium (1.25 mM) induced a significant proteolysis in membrane cytoskeletal proteins, compared with magnesium (p < 0.001). Actin exhibited the highest sensitivity to calcium-induced proteolysis (6.8 ± 0.3 vs. 5.3 ± 0.6, p < 0.001), while spectrin (39.9 ± 1.0 vs. 40.3 ± 2.0, p = 0.393) and band-6 (6.3 ± 0.3 vs. 6.8 ± 0.8, p = 0.191) were more resistant to proteolysis after incubation with calcium in the range of endoplasmic reticulum concentrations (250 µM). Aggregation of membrane cytoskeletal proteins was determined after centrifugation and was significantly higher after incubation with calcium ions compared with control, EDTA and magnesium solutions (p < 0.001). In a supra-physiologic range of 1.25–10 mM of calcium ions, there was a nearly perfect linear relationship between calcium concentration and aggregation of erythrocyte membrane cytoskeletal proteins (R ² = 0.971, p < 0.001). Our observation suggests a strong interaction between calcium ions and membrane cytoskeletal network. Cumulative effects of disrupted calcium homeostasis on cytoskeletal proteins need to be further investigated at extended periods of time in disease states.     

Improved methods for reducing calcium and magnesium concentrations in tissue culture medium: application to studies of lymphoblast proliferation in vitro.

We have compared several methods for reducing calcium and magnesium concentrations in tissue culture medium, with the objective of producing selective deficiency effects on the growth of mouse (L5178Y) and human (P1R) lymphoblasts. In experiments in which calcium- and magnesium- "free" McCoy's medium was supplemented with 15% horse or fetal calf serum, enough calcium and magnesium was provided by serum to support normal lymphoblast growth rate. Either dialysis or chelating-resin treatment of horse or fetal calf serum reduced calcium and magnesium contents approximately 100-fold. Use of dialyzed sera resulted in reduced growth rate, although in most cases the reduction in growth could be attributed to other effects of dialysis on serum, inasmuch as growth in those experiments was not restored to normal by the addition of calcium and magnesium to the medium. In contrast, the reduction of lymphoblast growth rate that occurred when resin-treated serum was used was always attributable to removal of calcium and magnesium, as normal growth always occurred in cultures to which calcium and magnesium were added. To demostrate a growth-inhibiting effect on either mouse or human lymphoblasts by severe reduction of either calcium or magnesium in the presence of normal amounts of the alternative cation, it was necessary to (a) expose McCoy's Ca-Mg-"free" medium to chelating-resin to reduce further the residual cation concentrations; (b) wash cells from stock cultures in a medium devoid of calcium and magnesium prior to inoculation into experimental cultures; (c) reduce the proportion of serum in the final medium from 15 to 5%; and (d) add 100 muM EGTA to cultures. Under these conditions, growth of both cell types was completely abolished in the presence of normal magnesium but in the absence of added calcium, and markedly reduced in the presence of normal calcium but in the absence of magnesium. These modifications did not compromise growth in cultures containing normal concentrations of both ions.