Technical Parameters of Plastics (Mercox Cl-2B and Various Methylmethacrylates) Used in Scanning Electron Microscopy of Vascular Corrosion Casts

The most frequently used resins for vascular corrosion casting Mercox Cl-2B, Mercox Cl-2B diluted with methylmethacrylate (MMA) monomer and various self prepared MMA and hydroxyproyl-methacrylate mixtures were tested with regard to their thermostability, shrinkage, viscosity and replication quality. It was found that tempering of the plastics improves their thermostability with the exception of Mercox Cl-2B and that shrinkage depends on the amount of monomers a resin contains. In detail; Mercox Cl-2B has the lowest shrinkage (8.018%) whereas a hydroxypropyl-methacrylate mixture possessed the highest (20.408%). But, on the other hand, viscosity decreases with the quantity of monomers. All resins tested were able to replicate structures of 260nm height but the resins\u27 quality of replication was found to be limited by the effects of shrinkage. Finally, a method to estimate the blood volume of organs and tissues with the help of vascular corrosion casts is given

Scanning Electron Microscopy of Vascular Corrosion Casts - Technique and Applications: Updated Review

The present paper states very briefly the main steps leading to the technique of scanning electron microscopy (SEM) of vascular corrosion casts. From the terms presently used (injection method, microcorrosion cast, injection replica, vascular corrosion cast, vascular cast) the use of vascular corrosion cast for lymphatic and blood vessels is recommended. Specification and pretreatment (kind, volume, dosage of anticoagulants, vasoactive substances and spasmolytica used) of the animals examined are referenced as they are available from the literature. The recommendation is given to pay more attention to these parameters than done so far. The steps necessary for producing reasonable and suitable vascular corrosion casts are critically described. Special attention is paid to the physical and chemical properties of the casting media and their significance for polymerization, shrinkage, casting quality, corrosion resistance, and thermal and spatial stability. Emphasis is also focused on the advantages of cutting the vascular corrosion casts embedded in an ice block by a band saw, a self constructed multi-blade cutting device or a mini wheel-saw placed in the chamber of a cryomicrotome. From the drying methods presently used freeze-drying is stressed because of minimal specimen damage. To render casts conductive in most cases sputter-coating is sufficient. It is recommended to run the SEM with 5 - 10 kV since the resolution received still reveals all details the casting media presently can replicate. Further the application of scanning electron microscopy of vascular corrosion casts in fully differentiated normal tissue, in pathologic tissue as well as in developing tissues and organs is stated. Lastly possibilities and conditions are discussed under which SEM of vascular corrosion casts can serve to quantify vascular structures in order to make the technique more than pure descriptive

Diluted and Undiluted Mercox Severely Destroy Unfixed Endothelial Cells. A Light and Electron Microscopic Study Using Cultured Endothelial Cells and Tadpole Tail Fin Vessels

Mercox is a methylmethacrylate-based resin which is widely used for vascular corrosion casting with subsequent scanning electron microscopic analysis. In the present study the effect of undiluted and diluted Mercox (4+1; volume + volume; Mercox: monomeric methylmethacrylate (MMA); 0.02 g catalyst MA/ml Mercox) and methylmethacrylate with and without catalyst MA (0.625 g/10 ml MMA) on fixed and unfixed endothelial cells was studied. Light microscopy (LM) of cultured capillary endothelial cells (ECs), which were replicated with diluted or undiluted Mercox shows degranulation and membrane perturbation of ECs, while no morphological changes occur in glutaraldehyde-prefixed ECs. Scanning electron microscopy (SEM) of replicas ( = resin blocks) polymerized on prefixed ECs reveals unchanged ECs and replicas show many details. Unfixed ECs are destroyed and replicas reveal aberrant features. Transmission electron microscopy (TEM) of prefixed and unfixed ECs (cultured endothelial cells, endothelial cells of perfusion prefixed and of unfixed tadpole tail fin vessels) substantiates LM and SEM findings. Prefixed ECs resist Mercox without fine structural changes, while unfixed cells undergo destruction. It is recommended to fix vessels prior to casting. Extravasations in microvessels are considered to be caused by focal chemical destruction of endothelial cells

Modulated by gasotransmitters: BK channels

© Springer-Verlag Berlin Heidelberg 2012. All rights are reserved. Calcium-activated potassium BK channels interconnect cellular activity, calcium signaling, and cell metabolism. Major virtues of these channels are their adaptability to different functions, their versatile physiology, and their capacity being modulated. The channels are present in a large variety of cells and organs in different forms of life from bacteria to men. Scientists attracted to these channels have produced a great wealth of information regarding their structure and function. Mutations at channels proteins are involved in a number of diseases (channelopathies), like diabetes, epilepsy, or heart failure. The gasotransmitters NO, CO, and H2S all act on BK channels directly or indirectly via signaling pathways. In this chapter, we will briefly summarize some of the basic properties of BK channels and focus on aspects of BK channel modulation by gasotransmitters and their implications in physiology and pathophyiology

Hydrogen sulfide increases calcium-activated potassium (BK) channel activity of rat pituitary tumor cells

Hydrogen sulfide (H2S) is the third gasotransmitter found to be produced endogenously in living cells to exert physiological functions. Large conductance (maxi) calcium-activated potassium channels (BK), which play an important role in the regulation of electrical activity in many cells, are targets of gasotransmitters. We examined the modulating action of H2S on BK channels from rat GH3 pituitary tumor cells using patch clamp techniques. Application of sodium hydrogen sulfide as H2S donor to the bath solution in whole cell experiments caused an increase of calcium-activated potassium outward currents. In single channel recordings, H2S increased BK channel activity in a concentration-dependent manner. Hydrogen sulfide induced a reversible increase in channel open probability in a voltagedependent, but calcium independent manner. The reducing agent, dithiothreitol, prevented the increase of open probability by H 2S, whereas, the oxidizing agent thimerosal increased channel open probability in the presence of H2S. Our data show that H2S augments BK channel activity, and this effect can be linked to its reducing action on sulfhydryl groups of the channel protein

Gasotransmitters: Physiology and pathophysiology

© Springer-Verlag Berlin Heidelberg 2012. All rights are reserved. Since the epochal discovery of the radical and highly toxic gas nitric oxide (NO) as a signaling molecule, two other no less toxic gases - carbon monoxide (CO) and hydrogen sulfide (H2S) - have been found to also be involved in a plethora of physiological and pathophysiological functions. The gases termed gasotransmitters play an increasingly important role in understanding how signalling into and between cells is modulated and fine-tuned. The advent of gasotransmitters has profoundly changed our way of thinking about biosynthesis, liberation, storage and action mechanisms in cellular signaling. In recent years an impressive amount of new data, distributed throughout the existing literature, has been generated. For this book the editors have recruited distinguished colleagues in the field to summarize and review important biological, pharmacological and medical functions and their implications, as well as methods for the detection of gasotransmitters

The Vascularization of the Skin of the Atlantic Hagfish, Myxine glutinosa L. as Revealed by Scanning Electron Microscopy of Vascular Corrosion Casts

The vascularization of three different (A, B, C) skin regions (from the level of the heart to the cloaca including dorsal, lateral and ventral skin areas) of the Atlantic hagfish, Myxine glutinosa L. was studied by scanning electron microscopy of vascular corrosion casts. Vessel variables were measured either from semithin sections (diameters) or from vascular corrosion casts (diameters, lengths) and total blood capacities as well as vessel surfaces per unit skin area (mm2) were calculated. There are no significant differences in the number of subepidermal capillary meshes (ranging from 164 to 185 meshes per micrograph) in areas A, B or C nor in vessel lengths. The average vessel length per mm2 is 32 mm. Assuming an average diameter of 22.3 μm these vessels have an average surface of 2.24 mm2 and a volume of 12.5 nanoliters (nl). In contrary weighing two pieces ( 5 mm times 5 mm in size) of the whole skin vascular bed - knowing the density of the casting medium -results in only one fifth of that volume. Overestimation of vessel lengths and diameters by measuring casted structures from micrographs on the one hand and inaccuracies in weighing or dissection of casted skin pieces on the other hand are discussed as sources of observed differences

Phosphorylation of BK channels modulates the sensitivity to hydrogen sulfide (H2S)

© 2014 Sitdikova, Fuchs, Kainz, Weiger and Hermann. Introduction: Gases, such as nitric oxide (NO), carbon monoxide (CO), or hydrogen sulfide (H2S), termed gasotransmitters, play an increasingly important role in understanding of how electrical signaling of cells is modulated. H2S is well-known to act on various ion channels and receptors. In a previous study we reported that H2S increased calcium-activated potassium (BK) channel activity. Aims: The goal of the present study is to investigate the modulatory effect of BK channel phosphorylation on the action of H2S on the channel as well as to recalculate and determine the H2S concentrations in aqueous sodium hydrogen sulfide (NaHS) solutions. Methods: Single channel recordings of GH3, GH4, and GH4 STREX cells were used to analyze channel open probability, amplitude, and open dwell times. H2S was measured with an anion selective electrode. Results: The concentration of H2S produced from NaHS was recalculated taking pH, temperature salinity of the perfusate, and evaporation of H2S into account. The results indicate that from a concentration of 300 μM NaHS, only 11-13%, i.e., 34-41 μM is effective as H2S in solution. GH3, GH4, and GH4 STREX cells respond differently to phosphorylation. BK channel open probability (Po) of all cells lines used was increased by H2S in ATP-containing solutions. PKA prevented the action of H2S on channel Po in GH4 and GH4 STREX, but not in GH3 cells. H2S, high significantly increased Po of all PKG pretreated cells. In the presence of PKC, which lowers channel activity, H2S increased channel Po of GH4 and GH4 STREX, but not those of GH3 cells. H2S increased open dwell times of GH3 cells in the absence of ATP significantly. A significant increase of dwell times with H2S was also observed in the presence of okadaic acid. Conclusions: Our results suggest that phosphorylation by PKG primes the channels for H2S activation and indicate that channel phosphorylation plays an important role in the response to H2S

Phosphorylation of BK channels modulates the sensitivity to hydrogen sulfide (H2S)

© 2014 Sitdikova, Fuchs, Kainz, Weiger and Hermann. Introduction: Gases, such as nitric oxide (NO), carbon monoxide (CO) or hydrogen sulfide (H2S), termed gasotransmitters, play an increasingly important role in understanding of how electrical signaling of cells is modulated. H2S is well known to act on various ion channels and receptors. In a previous study we reported that H2S increased calcium-activated potassium (BK) channel activity. Aims: The goal of the present study is to investigate the modulatory effect of BK channel phosphorylation on the action of H2S on the channel as well as to recalculate and determine the H2S concentrations in aqueous sodium hydrogen sulfide (NaHS) solutions. Methods: Single channel recordings of GH3, GH4 and GH4 STREX cells were used to analyze channel open probability, amplitude and open dwell times. H2S was measured with ananion selective electrode. Results: The concentration of H2S produced from NaHS was recalculated taking pH, temperature salinity of the perfusate and evaporation of H2S into account. The results indicate that from a concentration of 300 μM NaHS, only11-13%, i.e. 34-41 μM is effective as H2S in solution. GH3, GH4 and GH4 STREX cells respond differently to phosphorylation. BK channel open probability (Po) of all cells lines used was increased by H2S in ATP containing solutions. PKA prevented the action of H2S on channel Po in GH4 and GH4 STREX, but not in GH3 cells. H2S, high significantly increased Po of all PKG pretreated cells. In the presence of PKC, which lowers channel activity, H2S increased channel Po of GH4 and GH4 STREX, but not those of GH3 cells. H2S increased open dwell times of GH3 cells in the absence of ATP significantly. A significant increase of dwell times with H2S was also observed in the presence of okadaic acid. Conclusions: Our results suggest that phosphorylation by PKG primes the channels for H2S activation and indicate that channel phosphorylation plays an important role in the response to H2S

Hydrogen sulfide induces hyperpolarization and decreases the exocytosis of secretory granules of rat GH3 pituitary tumor cells

© 2015 Elsevier Inc. All rights reserved. The aim of the present study was to evaluate the effects of hydrogen sulfide (H2S) on the membrane potential, action potential discharge and exocytosis of secretory granules in neurosecretory pituitary tumor cells (GH3). The H2S donor - sodium hydrosulfide (NaHS) induced membrane hyperpolarization, followed by truncation of spontaneous electrical activity and decrease of the membrane resistance. The NaHS effect was dose-dependent with an EC50 of 152 μM (equals effective H2S of 16-19 μM). NaHS effects were not altered after inhibition of maxi conductance calcium-activated potassium (BK) channels by tetraethylammonium or paxilline, but were significantly reduced after inhibition or activation of ATP-dependent potassium channels (KATP) by glibenclamide or by diazoxide, respectively. In whole-cell recordings NaHS increased the amplitude of KATP currents, induced by hyperpolarizing pulses and subsequent application of glibenclamide decreased currents to control levels. Using the fluorescent dye FM 1-43 exocytosis of secretory granules was analyzed in basal and stimulated conditions (high K+ external solution). Prior application of NaHS decreased the fluorescence of the cell membrane in both conditions which links with activation of KATP currents (basal secretion) and activation of KATP currents and BK-currents (stimulated exocytosis). We suggest that H2S induces hyperpolarization of GH3 cells by activation of KATP channels which results in a truncation of spontaneous action potentials and a decrease of hormone release