Effects of serine/threonine protein phosphatases on ion channels in excitable membranes

This review deals with the influence of serine/threonine-specific protein phosphatases on the function of ion channels in the plasma membrane of excitable tissues. Particular focus is given to developments of the past decade. Most of the electrophysiological experiments have been performed with protein phosphatase inhibitors. Therefore, a synopsis is required incorporating issues from biochemistry, pharmacology, and electrophysiology. First, we summarize the structural and biochemical properties of protein phosphatase (types 1, 2A, 2B, 2C, and 3-7) catalytic subunits and their regulatory subunits. Then the available pharmacological tools (protein inhibitors, nonprotein inhibitors, and activators) are introduced. The use of these inhibitors is discussed based on their biochemical selectivity and a number of methodological caveats. The next section reviews the effects of these tools on various classes of ion channels (i.e., voltage-gated Ca(2+) and Na(+) channels, various K(+) channels, ligand-gated channels, and anion channels). We delineate in which cases a direct interaction between a protein phosphatase and a given channel has been proven and where a more complex regulation is likely involved. Finally, we present ideas for future research and possible pathophysiological implications.     

Morphological variability, lectin binding and Na+,K+-activated adenosine triphosphatase activity of isolated Müller (glial) cells from the rabbit retina

Rabbit retinal Müller cells were isolated by means of papaine and mechanical dissociation. These cells were shown to have a well preserved morphology and to preserve viability for many hours. Intense wheat germ agglutinin binding occurs on the photoreceptor side of Müller cells, especially in the microvillous region. Rabbit retinal Müller cells have a Na+,K+-activated adenosine triphosphatase activity in the same order of magnitude as brain astroglial cells.     

Copolymers with soft and hard segments based on 2,2-dimethyltrimethylene carbonate and ε-caprolactone

Thermoplastic elastomers with ABA-block structure, A representing the hard segment and B the soft segment, were prepared by ring-opening polymerization from 2,2-dimethyltrimethylene carbonate (DTC, 1 ) and ε-caprolactone (ECL, 2 ) as monomers. Typical anionic initiators, viz. the dilithium salt of hydroxytelechelic poly(ethylene oxide) 200 ( 4 ) and of polytetrahydrofuran 1000 ( 5 ) and as an insertion initiator the bis(diethylaluminium) salt of triethylene glycol were used. The soft block is formed by a sequence of random (or tapered) structure comprising equimolar amounts of the two monomers and obtained by simultaneous polymerization of DTC and ECL. The hard blocks consist of highly crystalline poly(2,2-dimethyltrimethylene carbonate) sequences. The thermal properties of the polymers depend on the sequence length, the content of heterodiads in the soft block and the thermal history of the samples. Copolymer films with short end blocks and high content of heterodiads show rubber-elastic properties at normal conditions.     

Quantitation of cytomegalovirus (hCMV) DNA and β-actin DNA by duplex real-time fluorescence PCR in solid organ (liver) transplant recipients

Even now rare human cytomegalovirus (hCMV) reactivation is still a life-threatening complication after solid organ transplantation. Although PCR techniques are regarded as the most sensitive detection methods for hCMV, their accuracy and reproducibility are limited. This is a major disadvantage with quantitative PCR assays, which are thought to provide valuable information about hCMV latency or active viral replication in transplant patients. To enhance the diagnostic safety of quantitative hCMV PCR, we developed a duplex real-time fluorescence PCR that is capable of quantifying hCMV DNA and beta-actin DNA as internal control simultaneously within one reaction. By the use of 6-carboxyfluorescein and hexa-chloro-6-carboxyfluorescein as reporter fluorophores and 4-(4'-dimethylamino-phenylazo) benzoic acid as dark quencher dye, hCMV DNA and beta-actin DNA could be quantified in parallel in a wide linear range from 10(1) to 10(7) copies, each. To test the clinical applicability of this approach, we investigated hCMV DNA kinetics in peripheral leukocytes of three hCMV antigen-positive and four antigen-negative patients after liver transplantation, as assessed by intracellular hCMV pp65 alkaline phosphate-anti-alkaline phosphate (APAAP) complex. While all APAAP-negative individuals remained PCR negative, kinetics of HCMV DNA in leukocyte DNA samples of APAAP-positive patients correlated closely with hCMV antigen tests. Here, comparison of separate and simultaneous target quantitation revealed identical results. It is of interest that, while single hCMV antigen positivity is commonly not regarded as a reliable parameter of viral reactivation, in our study a viral load greater than 10(4) copies/2x10(5) beta-actin DNA copies clearly indicated a subsequent increase in APAAP-positive leukocytes. We conclude that with the presented method the reliability of hCMV quantitation via real-time PCR can be substantially increased and may be used to monitor hCMV kinetics in vivo.     

Histamine excites GABAergic cells in the rat substantia nigra and ventral tegmental area in vitro

We have investigated the effect of histamine (HA) on spontaneous firing of dopaminergic (DA) and GABAergic neurons in the substantia nigra (SN) and the ventral tegmental area (VTA) of the rat in vitro. Single-unit extracellular recordings were obtained and drugs were bath applied. In both regions application of HA (10 and 100 μM) did not affect the firing frequency of DAergic cells, but increased the firing of GABAergic neurons. The histamine-induced excitation was blocked by the H₁ receptor antagonist mepyramine (1 μM), but was unaffected by application of the H₂ antagonist cimetidine (50 μM) or the H₃ antagonist thioperamide (10 μM). Our results suggest that histamine does not directly inhibit dopaminergic neurons in SN and VTA, but rather that this inhibition is mediated through histamine-induced excitation of GABAergic neurons.     

Observation of a nematic phase displayed by a polysiloxane with trinitrofluorenones as side groups

The synthesis and the results of the structural study of two copolysiloxanes with laterally fixed trinitrofluorenone (TNF) units is reported. The two copolysiloxanes having 2,4 ( 1a ) and 5,3 ( 1b ) dimethylsiloxane comonomer units per TNF side group differ significantly in their phase behaviour as evident from optical microscopy, differential scanning calorimetry and X-ray scattering: 1b shows a nematic mesophase whereas 1a is an amorphous material. The different phase behaviour is discussed in terms of microphase separation between the siloxane backbone and TNF side groups.     

Osteoprotegerin reduces the loss of periarticular bone mass in primary and secondary spongiosa but does not influence inflammation in rat antigen-induced arthritis

Objective: To assess the effect of osteoprotegerin (OPG) on joint swelling, synovial inflammation and cartilage destruction, periarticular and axial bone volume, and bone turnover in rat antigen-induced arthritis (AIA). Design: Rats were treated with OPG (3 mg/kg/day) at regular intervals from day 1 to day 20 of AIA. Disease activity was evaluated by measurement of joint swelling as well as, joint inflammation and destruction by histology. Bone volume and cellular turnover parameters of secondary spongiosa of the right tibia head and the third lumbar vertebra were evaluated by histomorphometry. Periarticular bone volume of the primary spongiosa at the right tibia head was measured by linear scanning. The findings were compared with those of PBS-treated AIA and healthy animals. Result: OPG treatment did not reduce joint swelling or histological signs of inflammation. Cartilage destruction was reduced. However, this effect did not reach statistical significance . In the secondary spongiosa OPG treatment reduced the loss of periarticular bone volume. However, the latter did not reach the level of healthy controls. OPG treatment significantly reduced parameters of bone formation and bone resorption. In the primary spongiosa, OPG-treatment led to a higher amount of mineralized tissue and a greater number of trabeculae compared to PBS-treated animals with AIA or healthy controls. In the axial skeleton, OPG treatment reduced bone formation and bone resorption parameters compared to healthy animals. This treatment had no influence on bone volume. Conclusions: In periarticular bone of AIA rats, OPG treatment reduced the loss of bone volume and decreased the bone turnover, thus preventing periarticular bone destruction. OPG treatment had no influence on inflammatory process or on cartilage destruction. Received 2 June 2005; returned for revision 26 July 2005; returned for final revision 9 August 2005; accepted by M. Parnham 24 September 2005 Presented in part at the 66. Annual Meeting of the American College of Rheumatology, New Orleans, U.S.A., October 2002, and at the 25. Annual Meeting of the American Society of Bone and Mineral Research, Minneapolis, USA, September 2003 Supported by grants from the Thuringian Ministry of Science, Research and Art (B307-01025, B378-01017), the Interdisciplinary Center for Clinical Research (IZKF) Jena, and the Deutsche Forschungsgemeinschaft (Br 1372/5-1) Osteoprotegerin was generously provided by Amgen (Thousand Oaks, CA, USA). Drs. Neumann and Oelzner contributed equally to this work.     

Sequential expression of functions during macrophage differentiation in murine bone marrow liquid cultures

In the presence of a colony-stimulating factor, murine bone marrow cells proliferate and differentiate into macrophages. This culture system was taken as a model to study the expression of various functions by macrophages in the course of maturation. Several tests were performed daily and in parallel from the same batch of cells. It was found that certain functions were expressed early and were also characteristic for mature macrophages such as Fc receptors, phagocytosis of latex beads and unspecific esterase activity. Other functions appeared and disappeared in an ordered sequence, such as the response to macrophage migration inhibitory factor and chemotactic factor as well as the production of interferon and of plasminogen activator. The time course of functional expression was strongly dependent on proliferation of precursor cells as well as proliferation of differentiated macrophages. It is suggested that the phenotypic expression of functions during differentiation is the basis for the functional heterogeneity of macrophages.