The Physical State of Potassium in Frog Skeletal Muscle Studied by Ion-Sensitive Microelectrodes and by Electron Microscopy: Interpretation of Seemingly Incompatible Results

According to the commonly accepted membrane pump theory most of cellular K+ ions are freely dissolved in free cellular water; the alternative association-induction hypothesis postulates that the bulk of cellular K+ is adsorbed (weakly bound) to cellular proteins which are maintained in a specific labile state in the cytoplasm of a living cell. K+ activities measured with ion-sensitive microelectrodes in the cytoplasm of frog skeletal muscle seem to confirm the claim that most of cellular K+ ions are free in cellular water. On the other hand, it is evident from electron microscopic ion binding studies that in frog skeletal muscle most of cellular K+ ions are adsorbed to cellular proteins. The conflicting results can be explained with the assumption that a damage of the cytoplasm caused by the impaling microelectrode leads to a liberation of adsorbed ions. The possibility that microelectrodes damage the muscle cytoplasm is tested by using the light microscope. It is found that microelectrodes produce visible traumas which increase with time. Electron microscopic ion binding studies with damaged muscle support the view that monovalent cations are liberated in the disturbed area of a muscle fiber. It is concluded that a K+-sensitive microelectrode is not suited to determine the concentration of free K+ ions in intact frog skeletal muscle

Optimal Freeze-Drying of Cryosections and Bulk Specimens for X-Ray Microanalysis

Electron microscopic investigations of rapidly frozen specimens of striated muscle, either frozen-hydrated or obtained after different dehydration procedures, have shown that the subcellular distribution of the main cellular cation K+ or its surrogates Rb+, Cs+, or Tl+ does not follow the water distribution but follows certain proteins. Conflicting results obtained by X-ray microanalysis of freeze-dried cryosections are explained by showing that freeze-drying of bulk specimens and cryosections must be carried out for rather long periods at low temperature in order to avoid severe shrinkage and ion redistribution artefacts. Proposals for future freeze-drying studies are derived from the concept that cellular water is organized differently from normal free water and that proteins of living cells are able to selectively adsorb alkali-metal ions

Low Temperature Embedding of Chemically Unfixed Biological Material After Cryosorption Freeze-Drying

After freeze-drying of bulk specimens in a newly developed cryosorption freeze-dryer (CFD) a special accessory is used to infiltrate the specimens in Lowicryl HM20 and to polymerize them at low temperature by ultra-violet (UV) irradiation within the CFD chamber in flat embedding moulds. The accessory allows polymerization in a dry, oxygen free environment without the risk of evaporation of volatile components of the resin which may lead to unsatisfactory polymerization. First results demonstrate the quality of structure preservation of biological material not treated with any chemical fixative

Freeze-Dried Embedded Specimens for Biological Microanalysis

The main problems associated with freeze-drying of biological material for electron microscopy concern the freeze-drying temperatures and times necessary to minimize artifacts. Due to the many parameters involved these problems have to be resolved experimentally. It can be shown that good morphological preservation of chemically unfixed material is possible when freeze-drying is done exclusively in a temperature range between -80°C and -50°C. OsO4 vapour fixation of the freeze-dried tissue is not necessary and should be avoided because it may cause ion redistribution artifacts. Embedding at low temperature of properly freeze-dried material does not seem to disturb structure and ion distribution of the freeze-dried material. Hence, sections of such freeze-dried material and embedded biological material seem to be suitable for microanalysis. Preliminary micro-analytical results obtained from sections of freeze-dried and Lowicryl K11M embedded muscle reveal an uneven distribution of potassium in the sarcomeres similar to the visualized uneven distribution of the electron dense thallium (potassium surrogate) in frozen hydrated cryosections. A comparison of different cryomethods shows that freeze-drying and embedding is the simplest way to obtain stable thin sections of chemically unfixed biological material which, for instance, may be used for future microanalytical investigation of the interaction of proteins with physiological and non-physiological ions

The Cell Water Problem Posed by Electron Microscopic Studies of Ion Binding in Muscle

The question whether cell K+ is free or bound in the striated frog muscle has been investigated during the last 10 years by different cryomethods and electron microscopy. The results support the view that most of cellular ions are osmotically inactive and that therefore the observed cell water activity must be explained by a model which assumes a specific cell water structure. According to the association-induction hypothesis, cell water is influenced by macromolecules and has low solubilities for Na+ and other solutes which therefore are partly excluded from cellular water. Autoradiography of frozen hydrated Na+ loaded muscles and microanalytical studies with freeze-dried cryosections of ouabain treated muscle support the view that cell water has the proposed Na+ exclusion property. It is concluded that problems such as cell volume regulation and muscle contraction cannot be understood completely without taking into account cellular ion binding and a specific cell water structure; in addition, mainly due to these cell properties it seems to be impossible to avoid volume changes of cells and subcellular compartments during conventional chemical fixation and dehydration of biological specimens

Adsorption Staining of Freeze-Substituted and Low Temperature Embedded Frog Skeletal Muscle with Cesium: A New Method for the Investigation of Protein-Ion Interactions

A new adsorption staining method for transmission electron microscopy is described by means of which cellular adsorption sites of alkali-metal ions can be visualized in freeze-substituted and low temperature embedded biological material. The main features of this staining method are: 1) the use of Cs+ -ions which are known to accumulate in living cells like K+ -ions and 2) the removal of the staining solution from thin sections of the embedded material by centrifugal force. It is shown that sections of freeze-substituted and Lowicryl embedded frog skeletal muscle which has not been treated with chemical fixatives can be stained with electron-dense Cs+ -ions: protein sites of preferential ion adsorption are visualized. These sites are similar to those accumulating monovalent ions in living cells as had been shown previously with frozen-hydrated preparations. An observed pH-dependency of the adsorption staining is consistent with the view that the ion adsorption sites are β- and γ-carboxyl groups of cellular proteins. The results obtained so far indicate that the new method can be used to investigate weak interactions between cellular proteins and different ions by electron microscopic methods

Two Opposing Theories of the Cell: Experimental Testing by Cryomethods and Electron Microscopy

A main controversial issue in cell biology concerns the molecular mechanism responsible for K+ accumulation in living cells and Na+ exclusion from them. The alternative theoretical descriptions of these phenomena are based on different assumptions about the physical state of cellular Na+, K+ and H2O. In this article it is shown with striated muscles how cryomethods and microanlytical electron microscopy may be used to test the opposing theories. It is concluded that these methods may yield more realistic informations about the physical state of cellular K+ and Na+ than measurements with ion sensitive microelectrodes or the reference phase method of Horowitz and coworkers. The results obtained with different cryomethods, especially with autoradiography of frozen hydrated preparations and electron microscopy of frozen hydrated cryosections support the view that most of cellular K+ is bound to macromolecules and hence osmotically inactive. These findings suggest that problems of cell volume changes either in the living state or during preparative procedures (e.g. during chemical fixation) can only be understood by a model which takes into account properties of the cytoplasmic matrix and its associated water. Further experimental evidence for such a model is provided by electron micrographs obtained after glutaraldehyde fixation of normal and swollen muscles which are compared with results obtained after freeze-substitution

Freeze-Dried Human Leukocytes Stabilized with Uranyl Acetate During Low Temperature Embedding or with OsO4 Vapor After Embedding

Two new simple stabilization procedures for freeze-dried biological material are introduced which are compatible with low temperature embedding (LTE) in Lowicryl. The first method uses a Lowicryl K11M/HM20 mixture supplemented with 0.3% uranyl acetate for LTE. For the second method polymerized Lowicryl blocks containing the freeze-dried material are exposed to OsO4 vapor which penetrates into the Lowicryl block and stabilizes the embedded specimen. The quality of structural preservation is demonstrated with human leukocytes

The subdwarf B star SB 290 - A fast rotator on the extreme horizontal branch

Hot subdwarf B stars (sdBs) are evolved core helium-burning stars with very thin hydrogen envelopes. In order to form an sdB, the progenitor has to lose almost all of its hydrogen envelope right at the tip of the red giant branch. In close binary systems, mass transfer to the companion provides the extraordinary mass loss required for their formation. However, apparently single sdBs exist as well and their formation is unclear since decades. The merger of helium white dwarfs leading to an ignition of core helium-burning or the merger of a helium core and a low mass star during the common envelope phase have been proposed. Here we report the discovery of SB 290 as the first apparently single fast rotating sdB star located on the extreme horizontal branch indicating that those stars may form from mergers.Comment: 5 pages, 4 figures, A&A letters, accepte