Pharmacological properties of angiotensin II receptors in cultured rabbit gingival fibroblasts

Certain interspecific hybrids of the fish Xiphophorus spontaneously develop melanoma induced by the derepression of the Xmrk oncogene. Xmrk is a recent duplicate of an orthologue of the mammalian epidermal growth factor receptor gene Egfr. In addition to a specific overexpression in melanoma, amino-acid substitutions in the extracellular domain leading to ligand-independent dimerisation and constitutive autophosphorylation are responsible for the tumorigenic potential of Xmrk. The Xmrk receptor induces several signal transduction pathways mediating cell proliferation and resistance to apoptosis and initiating dedifferentiation. Moreover, Xmrk upregulates the expression of the secreted protein osteopontin, inducing an autocrine loop possibly allowing invasion and survival in the dermis as a first step in malignancy. Hence, Xmrk is able to induce pathways essential for a transformed phenotype. Some of these events are equivalent to those found downstream of the mammalian Egfr, but others have clearly evolved differently or are specific for pigment cells. Xmrk is potentially hazardous, nonessential and located in a very unstable genomic region. Nevertheless, Xmrk has been maintained under purifying selection in divergent Xiphophorus species. Hence, Xmrk has probably a beneficial function under certain conditions. The analysis of this function is a major challenge for future research in the Xiphophorus model.     

Dynamic aspects of alpha-actinin in cultured rabbit fibroblasts studied with immunofluorescence

In this work we study alpha-actinin sites in rabbit fibroblasts in culture. For this purpose we extracted alpha-actinin from rabbit striated skeletal muscle and produced the relative antibodies in sheep. Electrophoresis was performed, on PAA slab-gel and the immunodiffusion test on agarose slab-gel. Antibodies were used for direct and indirect immunofluorescence techniques by TRIC and FITC. We noticed that in young fibroblasts alpha-actinin is concentrated in perinuclear regions, while in adult fibroblasts it is scattered, more or less evenly in the cytoplasm and above all on the plates which link the cell membrane to the substractum. Both the direct immunofluorescence method with antibodies anti-alpha-actinin, marked by TRITC, and the indirect one with sheep IgG, marked by FITC, gave identical results.     

Ion conductance and selectivity of single calcium-activated potassium channels in cultured rat muscle

The conductance and selectivity of the Ca-activated K channel in cultured rat muscle was studied. Shifts in the reversal potential of single channel currents when various cations were substituted for Ki+ were used with the Goldman-Hodgkin-Katz equation to calculate relative permeabilities. The selectivity was Tl+ greater than K+ greater than Rb+ greater than NH4+, with permeability ratios of 1.2, 1.0, 0.67, and 0.11. Na+, Li+, and Cs+ were not measurably permeant, with permeabilities less than 0.05 that of K+. Currents with the various ions were typically less than expected on the basis of the permeability ratios, which suggests that the movement of an ion through the channel was not independent of the other ions present. For a fixed activity of Ko+ (77 mM), plots of single channel conductance vs. activity of Ki+ were described by a two-barrier model with a single saturable site. This observation, plus the finding that the permeability ratios of Rb+ and NH+4 to K+ did not change with ion concentration, is consistent with a channel that can contain a maximum of one ion at any time. The empirically determined dissociation constant for the single saturable site was 100 mM, and the maximum calculated conductance for symmetrical solutions of K+ was 640 pS. TEAi+ (tetraethylammonium ion) reduced single channel current amplitude in a voltage-dependent manner. This effect was accounted for by assuming voltage-dependent block by TEA+ (apparent dissociation constant of 60 mM at 0 mV) at a site located 26% of the distance across the membrane potential, starting at the inner side. TEAo+ was much more effective in reducing single channel currents, with an apparent dissociation constant of approximately 0.3 mM.     

Ion channels in asthma

Ion channels are specialized transmembrane proteins that permit the passive flow of ions following their electrochemical gradients. In the airways, ion channels participate in the production of epithelium-based hydroelectrolytic secretions and in the control of intracellular Ca(2+) levels that will ultimately activate almost all lung cells, either resident or circulating. Thus, ion channels have been the center of many studies aiming to understand asthma pathophysiological mechanisms or to identify therapeutic targets for better control of the disease. In this minireview, we focus on molecular, genetic, and animal model studies associating ion channels with asthma.     

Effect of an inhibitor of glucosylceramide synthesis on cultured rabbit skin fibroblasts

1-Phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), an effective inhibitor of UDP-glucose:ceramide glucosyltransferase, caused growth inhibition of cultured rabbit skin fibroblasts in a dose-dependent manner. At 50 microM both threo and erythro isomers of PDMP completely suppressed the cell growth. Major gangliosides of the fibroblasts, GM3 and GD3, were greatly reduced in amounts in the presence of threo-PDMP and accumulation of ceramides was observed. Surface labeling with galactose oxidase and [3H]NaBH4 demonstrated that neural glycosphingolipids with four or more sugars present on the surface of control cells were not detectable when the fibroblasts were grown in medium containing threo-PDMP. Metabolic labeling of cellular glycosphingolipids with [14C]-galactose showed reduced incorporation of radioactivity into gangliosides and neutral glycosphingolipids when threo-PDMP was present in the medium. In contrast, the erythro isomer of PDMP did not affect the biosynthesis of glycosphingolipids, a result suggesting that the inhibitory effect of erythro-PDMP on cell growth was due to a mechanism other than the inhibition of glucosyltransferase.     

Ion channels activated by L-glutamate and GABA in cultured cerebellar neurons of the rat

Glutamate and GABA-receptor channels were investigated in explants of rat cerebellum grown in cell culture. The patch-clamp technique was used to examine neurons under whole cell clamp and the properties of channels were derived by analysis of glutamate and GABA-evoked current noise. In addition, single channel currents activated by glutamate were recorded from isolated outside-out patches of membrane. We found evidence for at least two types of glutamate receptor-channels in cerebellar cells. Some neurons exhibited a channel of 50 pS conductance with a Lorentzian noise spectrum of 5.9 ms time constant. Single channels were readily resolved both in whole cell clamp and excised patches. Other neurons possessed low conductance channels which produced two component spectra. Estimates of the single channel conductance gave a value of about 140 fS. GABA channel noise obtained from these cells was also fitted by two component spectra which gave single channel conductance of 16 pS.     

The effect of vitreous humour on prostaglandin production by cultured rabbit chorioretinal fibroblasts

Factors in vitreous humour which regulate prostaglandin production were investigated using cultured rabbit chorioretinal fibroblasts. These cells produced predominantly prostaglandin E2, 6-ketoprostaglandin F1 alpha, a compound likely to be a metabolite of prostaglandin E2 and 5-hydroxyeicosatetraenoic acid. The synthesis of 6-ketoprostaglandin F1 alpha was nearly completely inhibited by the cyclooxygenase inhibitor aspirin and partially inhibited by 10(-6) M dexamethasone (49%) and 10(-5) M forskolin (68%). Addition of 10% rabbit vitreous humour to subconfluent cells maintained in Dulbecco's modified Eagle's medium plus 1% fetal bovine serum resulted in stimulation of 6-ketoprostaglandin F1 alpha production by as much as 246% as measured by radioimmunoassay. Chorioretinal fibroblasts labelled by [3H]arachidonic acid incorporation into cellular phospholipids synthesised greater amounts of all labelled arachidonic acid metabolites in response to vitreous humour. It was concluded, therefore, that there are factors present in vitreous humour of molecular weight above 10 kDa which are capable of stimulating cellular cyclooxygenase activity. Confluent cells also responded to a factor(s) present in vitreous humour. The fraction of less than 10 kDa inhibited 6-ketoprostaglandin F1 alpha production by 50% when used at a concentration of 10%. Furthermore, 6-ketoprostaglandin F1 alpha production in confluent cells (but not subconfluent cells) was inhibited to 40% of control levels by vitamin C at a concentration of 1 mg/100 ml. The latter result points to an inhibitory role for vitamin C in vitreous humour. We conclude, therefore, that vitreous humour contains factors important for the regulation of prostaglandin metabolism in the eye.     

Ion channels in neuronal survival

The study of ion channels represents one of the most active fields in neuroscience research in China.In the last 10 years,active research in various Chinese neuroscience institutions has sought to understand the mechanisms responsible for sensory processing,neural development and neurogenesis,neural plasticity,as well as pathogenesis.In addition,extensive studies have been directed to measure ion channel activity,structure-function relationships,as well as many other biophysical and biochemical properties.T...     

Ion channels in nerve ending

The modern data about the structure and function of the nerve ending ion channels are generalized and systematized. Ion channels of nerve endings provide the forming of the rest membrane potential, excitability, generation of action potential, regulate the intracellular concentration of calcium ions, take part in exocytosis of synaptic vesicules, participate in short-term and long-term synaptic plasticity, ensure the modulation of presynaptic functions. Methods of investigation of ion channels and data about their localization in central and peripheral nerve systems are represented. The review gives the functional characteristics, molecular structure and mechanisms of regulation of the known voltage- and ligand-dependent ion channels, the role of the certain types of ion channels in the machinery of transmitter release.     

Ion channels in postnatal neurogenesis

Neural stem and progenitor cells (NSC/NPCs) are unspecialized cells found in the adult peri-ventricular and sub-granular zones that are capable of self-renewal, migration, and differentiation into new neurons through the remarkable process of postnatal neurogenesis. We are now beginning to understand that the concerted action of ion channels, multi-pass transmembrane proteins that allow passage of ions across otherwise impermeable cellular membranes tightly regulate this process. Specific ion channels control proliferation, differentiation and survival. Furthermore, they have the potential to be highly selective drug targets due to their complex structures. As such, these proteins represent intriguing prospects for control and optimization of postnatal neurogenesis for neural regeneration following brain injury or disease. Here, we concentrate on ion channels identified in adult ventricular zone NSC/NPCs that have been found to influence the stages of neurogenesis. Finally, we outline the potential of these channels to elicit repair, and highlight the outstanding challenges.     

Ion channels in autoimmune neurodegeneration

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system characterized by widespread inflammation, focal demyelination and a variable degree of axonal and neuronal loss. Ionic conductances regulate T cell activation as well as neuronal function and thus have been found to play a crucial role in MS pathogenesis. Since present therapeutical approaches are only partially effective so far, ion channel modulation as a future strategy was brought into focus. Here, we review the status quo concerning recent findings from ion channel research in MS and its animal model, experimental autoimmune encephalomyelitis.     

Ion selectivity in potassium channels

Potassium channels are tetrameric membrane-spanning proteins that provide a selective pore for the conduction of K(+) across the cell membranes. One of the main physiological functions of potassium channels is efficient and very selective transport of K(+) ions through the membrane to the cell. Classical views of ion selectivity are summarized within a historical perspective, and contrasted with the molecular dynamics (MD) simulations free energy perturbation (FEP) performed on the basis of the crystallographic structure of the KcsA phospholipid membrane. The results show that the KcsA channel does not select for K(+) ions by providing a binding site of an appropriate (fixed) cavity size. Rather, selectivity for K(+) arises directly from the intrinsic local physical properties of the ligands coordinating the cation in the binding site, and is a robust feature of a pore symmetrically lined by backbone carbonyl groups. Further analysis reveals that it is the interplay between the attractive ion-ligand (favoring smaller cation) and repulsive ligand-ligand interactions (favoring larger cations) that is the basic element governing Na(+)/K(+) selectivity in flexible protein binding sites. Because the number and the type of ligands coordinating an ion directly modulate such local interactions, this provides a potent molecular mechanism to achieve and maintain a high selectivity in protein binding sites despite a significant conformational flexibility.     

离子通道与肿瘤

钾、钙、氯等离子通道在肿瘤细胞中异常表达,与肿瘤的发生发展密切相关。其可能机制是离子通道通过调节细胞膜电位、细胞周期、细胞体积、胞内钙浓度和胞质pH值等调控肿瘤细胞增殖与凋亡。本文综述了离子通道与肿瘤关系的研究进展,随着研究不断深入,离子通道有可能成为防治肿瘤的新靶标。     

Phosphorylation of Ion channels

The introduction of highly specific reagents such as enzymes and inhibitors directly into living cells has proven to be a powerful tool in studying the modulation of cellular activity by protein phosphorylation. The use of exogenous kinases can be thought of as a pharmacological approach: this demonstrates that phosphorylation can produce modulation, but does not address the question of whether the cell actually uses this mechanism under normal physiological conditions. The complementary approach, the introduction of highly specific inhibitors such as R subunit or PKI, does ask whether endogenous kinase activity is necessary for a given physiological response. Together these two approaches have provided rather compelling evidence that cAMP-dependent and calcium/phospholipid-dependent protein phosphorylations can regulate membrane excitability. In several cases single-channel analysis has allowed the demonstration that an ion channel itself or something very close to the channel is the phosphorylation target, and it seems reasonable to assume that this will also be the case for many if not all of the other systems described above. Have any general principles emerged from the results to date? Certainly it seems clear that protein phosphorylation regulates not one but many classes of ion channels. As summarized in the Table, different channels can be modulated in different cells, some channels are activated while others are inhibited, and in some cells more than one channel is subject to modulation by phosphorylation. The list in the Table is probably not yet complete, and indeed it is not inconceivable that all ion channels can under appropriate conditions be regulated by phosphorylation. What aspect of channel function is altered by phosphorylation? The total membrane current, I, carried by a particular species of ion channel is given by Npi, where N is the number of active channels in the membrane, p is the probability that an individual channel will be open, and i is the single-channel current. In principle a change in I, the quantity measured in whole cell experiments, could be caused by a change in any one (or more) of the parameters, N, p or i (see Fig. 1). In the two cases in which single-channel measurements have allowed this question to be investigated, changes in N (Shuster et al., 1985) and p (Ewald et al., 1985) have been observed. Here again it seems unlikely that any one mechanism operates in all cases, and it would not be surprising to find that phosphorylation of some other channel results in a change in i.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ion channels and transporters in cancer. 1. Ion channels and cell proliferation in cancer

Progress through the cell mitotic cycle requires precise timing of the intrinsic molecular steps and tight coordination with the environmental signals that maintain a cell into the proper physiological context. Because of their great functional flexibility, ion channels coordinate the upstream and downstream signals that converge on the cell cycle machinery. Both voltage- and ligand-gated channels have been implicated in the control of different cell cycle checkpoints in normal as well as neoplastic cells. Ion channels mediate the calcium signals that punctuate the mitotic process, the cell volume oscillations typical of cycling cells, and the exocytosis of autocrine or angiogenetic factors. Other functions of ion channels in proliferation are still matter of debate. These may or may not depend on ion transport, as the channel proteins can form macromolecular complexes with growth factor and cell adhesion receptors. Direct conformational coupling with the cytoplasmic regulatory proteins is also possible. Derangement or relaxed control of the above processes can promote neoplasia. Specific types of ion channels have turned out to participate in the different stages of the tumor progression, in which cell heterogeneity is increased by the selection of malignant cell clones expressing the ion channel types that better support unrestrained growth. However, a comprehensive mechanistic picture of the functional relations between ion channels and cell proliferation is yet not available, partly because of the considerable experimental challenges offered by studying these processes in living mammalian cells. No doubt, such studies will constitute one of the most fruitful research fields for the next generation of cell physiologists.