Hubert R. Catchpole

The Nature of Ground Substance of Connective Tissue

In 1949 we proposed a relatively simple hypothesis concerning the nature of the ground substance of connective tissue. Since then we have continued working on this problem independently along different lines. One of us (H. R. C.) with his colleagues has been working with electrometrical and chemical methods toward a better understanding of ground substance as a whole, considered as a physicochemical system. The other (I. G.) with his students has aimed at getting a closer view of the submicroscopic structure and relations of ground substance with the electron microscope. Now after ten years, our separate paths have converged. We feel that it may be profitable to attempt to integrate our newer findings with the earlier ones in order to develop, extend, and remodel the original hypothesis. The emphasis in this essay is placed on presenting a unified point of view, insofar as possible, which extends through our actual findings, interpretations, and speculations. Only enough data are given to clarify this point of view, and the references to the original literature are accordingly limited. Many others could readily be cited, for and against the point of view we hold, and numerous other references could be collected in support ofother outlooks. In other words, this essay is designed to emphasize one particular way of looking at certain problems centering around the role of the ground substance of connective tissues; it is not intended as a summary of the literature in the field. Ten years ago we considered the ground substance of connective tissue to be the non-fibrillar, optically homogeneous, extra(or inter-) cellular material (1). This was stainable under certain conditions in sections with the periodic acid leukofuchsin stain (PAS) or metachromatically with

Interaction of ions and connective tissue.

The interaction of ions with tissues was studied by measuring liquid junction potentials between tissues and 0.15 M KCl, and LiCl, or 0.075 M CaCl2 and MgCl2, as well as with one tenth isotonic NaCl. For baseline values, 0.15 M NaCl was used. In this way a series of substitution and dilution potentials was established. As a connective tissue which varies in physicochemical state, use was made of the symphysis pubis of normal and castrate guinea pigs, and of guinea pigs in various stages of pregnancy. The former have tight, unrelaxed symphyses, while the latter show varying grades of relaxation. Experimentally, relaxation was also induced by the use of estrogens and relaxin in castrate guinea pigs. Unrelaxed symphyses showed high positive dilution potentials (+20 to + 30 mv), while highly relaxed symphyses showed zero or negative dilution potentials, approaching in some cases values calculated for aqueous salt junctions (—12.3 mv). These results are interpreted on the basis of a high concentration of negatively charged immobile colloid in tight connective tissue; following induction of the physiological change of relaxation, the potentials indicate low densities of immobile charged colloid. An estimate of 1250 for the average base binding equivalent weight of tissue colloids indicates a magnitude characteristic of mucopolysaccharides or mucoproteins. Evidence from other work indicates that relaxation in the symphosis is accompanied by a disaggregation (depolymerization) of components of ground substance, and by an increase in water soluble fractions. In relaxed symphyses, where colloid density is low, mobility data indicate that the cations approach their behaviour in water. In tight symphyses, selective effects appear, related to the high concentrations of immobile charge. Potassium in particular showed a marked decrease of mobility to approximately one-half its value in water. Effects with other ions were much smaller. Thus connective tissue in a state of high aggregation appears to act selectively as an ion-exchange resin for potassium; with breakdown of aggregates and uptake of water, selectivity is lost. From the NaCl dilution potentials, concentrations of immobile charges were estimated. Values ranged from 0.20 equivalents per liter in the tight symphysis to 0.05 equivalents per liter or less in the relaxed state. Values of this order of magnitude have been calculated from independent data.

A microprobe analysis of inorganic elements in Halobacterium salinarum

Halobacterium salinarum were grown on peptone agar containing 4.28 M NaCl, 0.036 M K and other salts. Stationary phase organisms were lifted onto carbon planchets, freeze‐dried, carbon coated and examined in a scanning electron microscope equipped with an X‐ray spectrometer. Intracellular element concentrations (mol/kg H2O) were determined using a bulk analysis program with appropriate standards. The cell K concentration was 110 times that of the medium. For Na this value was 0.3 and for Cl, 1.1. When Rb was present in the medium, its intracellular concentration was 77 times higher than the external value. The cation minus anion value suggests a high fixed negative charge, 0.72 equivalents. Intracellular apparent dielectric constants were calculated using cellular EMFs derived from the literature, and sodium concentration. The determined values ranged from 22–28 (vs 80 for normal water) suggesting phases of structured cell water. Ionic distributions in these extremophiles are treated according to the classical principles elucidated by Willard Gibbs and represents a heterogeneous system in thermodynamic equilibrium with the hypersaline environment. Factors to be considered are: (1) composition of Halobacterium and its immobile negative charge; (2) the physicochemical properties of the individual ions (charge, ionic radius, hydration energy, standard chemical potential); (3) the dielectric constant of the dispersion medium (water); and (4) the binding of ions, particularly potassium.

SOLUBILITY PROPERTIES OF SOME COMPONENTS OF THE GROUND SUBSTANCE IN RELATION TO INTRAVITAL STAINING OF CONNECTIVE TISSUE

The ground substance arid basement membranes of connective tissues were studied in tissues prepared by the Altmann-Gershl freezing-drying technique and stained by the McManus-Hotchkiss per-iodic acid-leucofuchsin method.2. 3 , After these procedures, the polysaccharide component or components of these structures are stained pink or red. The chemical basis and specificity of the method has been discussed by Hotchkiss,4 and it is apparent that free and bound lipids may he removed as possible sources of confusion, by methods rerommended by Hack.5 Ground substance and basement membrane are regarded as closely related entities in every way. They form an extracellular, optically homogeneous medium possessing a fluid to a gel-like consistency, whose most characteristic components are glycoproteins stained red by the McManus-Hotchkiss procedure. There is evidence that they are structurally organized on a submicroscopic level and that this niay vary with age, activity, and pathologic state. The components of the ground substance infiltrate and enclose a network of oriented fibrils and fibers6 The reactive material of the ground substance is normally remarkably insoluble when tested with numerous reagents: e.g., buffers from pH 3.6-11.2, ammonium sulfide, dilute ammonium, and sodium hydroxide and common organic reagents like ethanol, pyridine, amyl acetate, and methanol-chloroform. It is removed by pepsin, pangestin, trypsin, collagenase, and by the toxin of Clostridium welchii. For the purposes of this discussion, glycoprotein of normal ground substance will be briefly characterized as alcoholand water-insoluble. When the intravital dye, Evans blue, is injected intravenously into normal animals, and the tissues are fixed appropriately ten minutes later, dye appears in the connective tissue generally in such small amounts that the ground substance is practically unstained, or in specific regions only faintly stained. In the region of a rapidly growing transplantable mouse tumor (Earle HGW), the connective tissue was observed to be easily dissectable. Fixed and examined by the methods described, the ground substance of the connective tissue adjacent to the tumor and within it was found to be alcoholinsoluble but water-soluble. Staining with the per-iodic acid-leucofuchsin reagent was more intense in these areas. Further, when Evans blue was injected intravenously into these animals, sites corresponding to the presence of water-soluble stainable material in the region of and within the tumor were found to be stained intensely blue. Assuming that one component, a t least, of the connective tissue is a highly polymerized mucopolysaccharide or glycoprotein which, under certain conditions, becomes partially depoly-

A microprobe study of element distribution in vaginal epithelial cells of the rat

Microprobe analysis of vaginal epithelial cells shed during the estrous cycle of the rat was done to determine cellular elements present in successive stages: pro‐estrus, estrus, and post‐estrus. Smears of vaginal contents were placed on carbon planchettes, fixed by freeze‐drying, and examined in a scanning microscope with an energy dispersive spectrometer. Concentrations of Na, Mg, P, S, Cl, K, and Ca were calculated (mmol/kg dry weight) and analyzed statistically. For phosphorus a significant fall at estrus correlates with a loss of nuclear and cytoplasmic nucleic acids and nucleoproteins. An increase in sulfur at estrus is consistent with an accumulation of keratins over pro‐estrus and a greater increase over the post‐estrus epithelial cells. The epithelial cells of pro‐estrus are highest in Mg and Ca. By post‐estrus, the cells have recovered their Mg, not Ca. Potassium concentrations exhibited no significant change between the successive stages.

Effects of Temperature and Anions on Titration Curves of Frog Muscle

Titration curves of frog muscle at 2 �C and 25 �C were determined in vivo. Lowered temperature decreases respiration without change of colloidal charge or of ion distribution as estimated from charge. Anions which are inhibitors or metabolites combine with muscle colloids, changing the distribution of other ainions and cations.