Effects of NS1608 on MaxiK Channels in Smooth Muscle Cells from Urinary Bladder

Using the patch-clamp technique, we have characterized membrane currents in single detrusor smooth muscle cells from rat and human urinary bladder. From the voltage- and Ca²⁺-dependence of the current as well as the single channel conductance we conclude that rat and human urinary bladder smooth muscle cells express MaxiK channels. In smooth muscle cells from rat urinary bladder we tested the action of NS1608 on current through these MaxiK channels. Application of 10 μm NS1608 increased the amplitude of the current and this increase could be explained by a shift in the activation voltage of the MaxiK channels ∼100 mV towards more negative potentials. Charybdotoxin as well as paxilline, well known blockers of MaxiK channels, were able to reduce current through MaxiK channels in our cell preparation. In addition, application of 10 μm NS1608 hyperpolarized the membrane potential of the investigated cells. This hyperpolarization could be antagonized by the application of paxilline. We conclude that application of NS1608 results in the opening of MaxiK channels under physiological conditions that leads to a hyperpolarization of the cells. This hyperpolarization in turn could relax urinary bladder smooth muscle cells. MaxiK channels in these cells could therefore play a role in directly controlling muscle tone by regulating the membrane potential. This opens up the possibility of MaxiK channels being targets for the treatment of urge incontinence. Received: 19 July/Revised: 20 September 1999     

Molecular Mechanisms of Polymyxin B-Membrane Interactions: Direct Correlation Between Surface Charge Density and Self-Promoted Transport

We have studied the interaction of the polycationic peptide antibiotic polymyxin B (PMB) with asymmetric planar bilayer membranes via electrical measurements. The bilayers were of different compositions, including those of the lipid matrices of the outer membranes of various species of Gram-negative bacteria. One leaflet, representing the bacterial inner leaflet, consisted of a phospholipid mixture (PL; phosphatidylethanolamine, -glycerol, and diphosphatidylglycerol in a molar ratio of 81:17:2). The other (outer) leaflet consisted either of lipopolysaccharide (LPS) from deep rough mutants of PMB-sensitive (Escherichia coli F515) or -resistant strains (Proteus mirabilis R45), glycosphingolipid (GSL-1) from Sphingomonas paucimobilis IAM 12576, or phospholipids (phosphatidylglycerol, diphytanoylphosphatidylcholine). In all membrane systems, the addition of PMB to the outer leaflet led to the induction of current fluctuations due to transient membrane lesions. The minimal PMB concentration required for the induction of the lesions and their size correlated with the charge of the lipid molecules. In the membrane system resembling the lipid matrix of a PMB-sensitive strain (F515 LPS/PL), the diameters of the lesions were large enough (d= 2.4 nm ± 8%) to allow PMB molecules to permeate (self-promoted transport), but in all other systems they were too small. A comparison of these phenomena with membrane effects induced by detergents (dodecyltriphenylphosphonium bromide, dodecyltrimethylammonium bromide, sodiumdodecylsulfate) revealed a detergent-like mechanism of the PMB-membrane interaction. Received: 16 September 1997/Revised: 25 November 1997     

Membrane Potassium Channels and Human Bladder Tumor Cells: II. Growth Properties

These experiments were done to determine the effect of glibenclamide and diazoxide on the growth of human bladder carcinoma (HTB-9) cells in vitro. Cell growth was assayed by cell counts, protein accumulation, and ³H-thymidine uptake. Glibenclamide added at 75 and 150 μm for 48 hr reduced cell proliferation. Dose-inhibition curves showed that glibenclamide added for 48 hr reduced cell growth at concentrations as low as 1 μm (IC₅₀= 73 μm) when growth was assayed in the absence of added serum. This μM-effect on cell growth was in agreement with the dose range in which glibenclamide decreased open probability of membrane K_ATP channels. Addition of glibenclamide for 48 hr also altered the distribution of cells within stages of the cell cycle as determined by flow cytometry using 10⁻⁵ m bromodeoxyuridine. Glibenclamide (100 μm) increased the percentage of cells in G₀/G₁ from 33.6% (vehicle control) to 38.3% (P < 0.05), and it reduced the percentage of cells in S phase from 38.3% to 30.6%. On the other hand, diazoxide, which opens membrane K_ATP channels in HTB-9 cells, stimulated growth measured by protein accumulation, but it did not increase the cell number. We conclude that the sulfonylurea receptor and the corresponding membrane K_ATP channel are involved in mechanisms controlling HTB-9 cell growth. However, K_ATP is not rate-limiting among the signaling mechanisms or molecular switches that regulate the cell cycle. Received: 12 June 1997/Revised: 21 October 1997     

Kinetic Analysis of the Inhibitory Effect of Glibenclamide on KATP Channels of Mammalian Skeletal Muscle

We investigated the block of K_ATP channels by glibenclamide in inside-out membrane patches of rat flexor digitorum brevis muscle. (1) We found that glibenclamide inhibited K_ATP channels with an apparent K _i of 63 nm and a Hill coefficient of 0.85. The inhibition of K_ATP channels by glibenclamide was unaffected by internal Mg²⁺. (2) Glibenclamide altered all kinetic parameters measured; mean open time and burst length were reduced, whereas mean closed time was increased. (3) By making the assumption that binding of glibenclamide to the sulphonylurea receptor (SUR) leads to channel closure, we have used the relation between mean open time, glibenclamide concentration and K _D to estimate binding and unbinding rate constants. We found an apparent rate constant for glibenclamide binding of 9.9 × 10⁷ m ⁻¹ sec⁻¹ and an unbinding rate of 6.26 sec⁻¹. (4) Glibenclamide is a lipophilic molecule and is likely to act on sulfonylurea receptors from within the hydrophobic phase of the cell membrane. The glibenclamide concentration within this phase will be greater than that in the aqueous solution and we have taken this into account to estimate a true binding rate constant of 1.66 × 10⁶ m ⁻¹ sec⁻¹. Received: 7 July 1996/Revised: 4 October 1996     

Nonselective Cation and BK Channels in Apical Membrane of Outer Sulcus Epithelial Cells

The outer sulcus epithelium was recently shown to absorb cations from the lumen of the gerbil cochlea. Patch clamp recordings of excised apical membrane were made to investigate ion channels that participate in this reabsorptive flux. Three types of channel were observed: (i) a nonselective cation (NSC) channel, (ii) a BK (large conductance, maxi K or K _Ca ) channel and (iii) a small K⁺ channel which could not be fully characterized. The NSC channel found in excised insideout patch recordings displayed a linear current-voltage (I-V) relationship (27 pS) and was equally conductive for Na⁺ and K⁺, but not permeable to Cl⁻ or N-methyl-d-glucamine. Channel activity required the presence of Ca²⁺ at the cytosolic face, but was detected at Ca²⁺ concentrations as low as 10⁻⁷ m (open probability (P _o ) = 0.11 ± 0.03, n= 8). Gadolinium decreased P _o of the NSC channel from both the external and cytosolic side (IC₅₀∼ 0.6 μm). NSC currents were decreased by amiloride (10 μm− 1 mm) and flufenamic acid (0.1 mm). The BK channel was also frequently (38%) observed in excised patches. In symmetrical 150 mm KCl conditions, the I-V relationship was linear with a conductance of 268 pS. The Goldman-Hodgkin-Katz equation for current carried solely by K⁺ could be fitted to the I-V relationship in asymmetrical K⁺ and Na⁺ solutions. The channel was impermeable to Cl⁻ and N-methyl-d-glucamine. P _o of the BK channel increased with depolarization of the membrane potential and with increasing cytosolic Ca²⁺. TEA (20 mm), charybdotoxin (100 nm) and Ba²⁺ (1 mm) but not amiloride (1 mm) reduced P _o from the extracellular side. In contrast, external flufenamic acid (100 μm) increased P _o and this effect was inhibited by charybdotoxin (100 nm). Flufenamic acid inhibited the inward short-circuit current measured by the vibrating probe and caused a transient outward current. We conclude that the NSC channel is Ca²⁺ activated, voltage-insensitive and involved in both constitutive K⁺ and Na⁺ reabsorption from endolymph while the BK channel might participate in the K⁺ pathway under stimulated conditions that produce an elevated intracellular Ca²⁺ or depolarized membrane potential. Received: 14 October 1999/Revised: 10 December 1999     

Tight Junction Dynamics: Oscillations and the Role of Protein Kinase C

The present study aimed to characterize the role of protein kinase C (PKC) on the dynamics of tight junction (TJ) opening and closing in the frog urinary bladder. The early events of TJ dynamics were evaluated by the fast Ca⁺⁺ switch assay (FCSA), which consisted in opening the TJs by removing basolateral Ca⁺⁺ ([Ca⁺⁺] _bl ), and closing them by returning [Ca⁺⁺] _bl to normal values. Changes in TJ permeability can be reliably gauged through changes of transepithelial electrical conductance (G) determined in the absence of apical Na⁺. The FCSA allows the appraisal of drugs and procedures acting upon the mechanism controlling the TJs. The time courses of TJ opening and closing in an FCSA were shown to follow single exponential time courses. PKC inhibition by H7 (100 μm) caused a reduction of the rate of junction opening in response to removing [Ca⁺⁺] _bl , without affecting junction closing, indicating that PKC is a key element in the control of TJ opening dynamics in this preparation. H7 at 250 μm almost completely inhibits TJ opening in response to basolateral Ca⁺⁺ withdrawal. Subsequent H7 removal caused a prompt inhibition release characterized by a sharp G increase which, however, once started cannot be stopped by H7 reintroduction, Ca⁺⁺ being necessary to allow TJ recovery. A step rise of apical Ca⁺⁺ concentration ([Ca⁺⁺] _ap ) causes a reduction of the rate of TJ opening in a FCSA, an effect that is believed to be mediated by apical Ca⁺⁺ entering the open TJs. The specific condition of having Ca⁺⁺ only in the apical solution and the TJs located midway between the Ca⁺⁺ source (apical solution) and the Ca⁺⁺-binding sites presumably located at the zonula adhaerens, might configure a situation in which a control feedback loop is set up. A rise of [Ca⁺⁺] _ap during the phase of G increase in an FCSA causes a transient recovery of G followed by a subsequent escape phase where G increases again. Oscillations of G also appear in response to a rise of apical Ca⁺⁺. Both escape and oscillations result from the properties of the TJ regulatory feedback loop. In conclusion, the present results indicate that PKC plays a key role in TJ opening in response to extracellular Ca⁺⁺ withdrawal without major effect on the reverse process. In addition, PKC inhibition by H7 not only prevents TJ opening in response to basolateral Ca⁺⁺ removal but induces a prompt blockade of TJ oscillations induced by apical Ca⁺⁺, oscillations which reappear again when H7 is removed. Received: 9 May 2000/Revised: 30 August 2000     

Reversible Electropermeabilization of Mammalian Cells by High-Intensity, Ultra-Short Pulses of Submicrosecond Duration

Mouse myeloma cells were electropermeabilized by single square-wave electric pulses with amplitudes of up to ∼150 kV/cm and durations of 10–100 nsec. The effects of the field intensity, pulse duration and medium conductivity on cell viability and field-induced uptake of molecules were analyzed by quantitative flow cytometry using the membrane-impermeable fluorescent dye propidium iodide as indicator molecule. Despite the extremely large field strengths, the majority of cells survived the exposure to ultra-short field pulses. The electrically induced dye uptake increased markedly with decreasing conductivity of the suspending medium. We assigned this phenomenon to the transient electrodeformation (stretching) force that assumes its maximum value if cells are suspended in low-conductivity media, i.e., if the external conductivity σ_e is smaller than that of the cytosol σ_i. The stretching force vanishes when σ_e is equal to or larger than σ_i. Due to their capability of delivering extremely large electric fields, the pulse power systems used here appear to be a promising tool for the electropermeabilization of very small cells and vesicles (including intracellular organelles, liposomes, etc.). Received: 15 May 2001/Revised: 20 July 2001     

A Basolateral Chloride Conductance in Rat Lingual Epithelium

We used Ussing chamber measurements and whole-cell recordings to characterize a chloride conductance in rat lingual epithelium. Niflumic acid (NFA) and flufenamic acid (FFA), nonsteroidal anti-inflammatory aromatic compounds known to inhibit Cl⁻ conductances in other tissues, reduced transepithelial short-circuit current (I _sc ) in the intact dorsal anterior rat tongue epithelium when added from the serosal side, and reduced whole-cell currents in rat fungiform taste cells. In both Ussing chamber and patch-clamp experiments, the effect of NFA was mimicked by replacement of bath Cl⁻ with methanesulfonate or gluconate. In low Cl⁻ bath solution, the effect of NFA on whole-cell current was reduced. Replacement of bath Ca²⁺ with Ba²⁺ reduced the whole-cell Cl⁻ current. We conclude that a Ca²⁺-activated Cl⁻ conductance is likely present in the basolateral membrane of the rat lingual epithelium, and is present in the taste receptor cells from fungiform papillae. Further experiments will be required to identify the role of this conductance in taste transduction. Received: 8 September 1997/Revised: 27 March 1998     

Regulation of Cation-selective Channels in Liver Cells

In liver cells, cation-selective channels are permeable to Ca²⁺ and have been postulated to represent a pathway for receptor-mediated Ca²⁺ influx. This study examines the mechanisms involved in the regulation of these channels in a model liver cell line. Using patch-clamp recording techniques, it is shown that channel open probability is a saturable function of cytosolic [Ca²⁺], with half-maximal opening at 660 nm. By contrast, channel opening is not affected by membrane voltage or cytosolic pH. In intact cells, reduction of cytosolic [Cl⁻], a physiological response to Ca²⁺-mobilizing hormones and cell swelling, is also associated with an increase in channel opening. Finally, channel opening is inhibited by intracellular ATP through a mechanism that does not involve ATP hydrolysis. These findings suggest that opening of cation-selective channels is coupled to the metabolic state of the cell and provides a positive feedback mechanism for regulation of receptor-mediated Na⁺ and Ca²⁺ influx. Received: 8 October 1996     

Evolutionary History of MHC Class I Genes in the Mammalian Order Perissodactyla

We carried out an analysis of partial sequences from expressed major histocompatibility complex (MHC) class I genes isolated from a range of equid species and more distantly related members of the mammalian order Perissodactyla. Phylogenetic analysis revealed a minimum of six groups, five of which contained genes and alleles that are found in equid species and one group specific to the rhinoceros. Four of the groups contained only one, or very few sequences, indicating the presence of relatively nonpolymorphic loci, while another group contained the majority of the equid sequences identified. These data suggest that a diversification of MHC genes took place after the split between the Equidae and the Rhinocerotidae yet before the speciation events within the genus Equus. Received: 17 November 1998 / Accepted: 7 April 1999     

Evolutionary History of B1 Retroposons in the Genus Mus

Short interspersed DNA elements (SINEs) amplify by retroposition either by (i) successive waves of amplification from one or a few evolving master genes or by (ii) the generation of new master genes that coexist with their progenitors. Individual, highly conserved, elements of the B1 SINE family were identified from the GenBank nucleotide database using various B1 subfamily consensus query sequences to determine their integration times into the mouse genome. A comparison of orthologous loci in various species of the genus Mus demonstrated that four subfamilies of B1 elements have been amplifying within the last 1–3 million years. Therefore, B1 sequences are generated by coexisting source genes. Additionally, three B1 subfamilies have been concurrently propagated during subspecies divergence and strain formation in Mus, indicating very recent activity of this retroposon family. The patterns of intra- and interspecies variations of orthologous loci demonstrate the usefulness of B1 integrations as a phylogenetic tool. A single inconsistency in the phylogenetic trends was depicted by the presence of a B1 insert in an orthologous locus exclusively in M. musculus and M. pahari. However, DNA sequence analysis revealed that these were independent integrations at the same genomic site. One highly conserved B1 element that integrated at least 4–6 million years ago suggests the possibility of occasional function for B1 integrations. Received: 25 February 2000 / Accepted: 5 June 2000     

Reconstructing the Complex Evolutionary History of Hepatitis B Virus

A detailed analysis of the evolutionary history of hepatitis B virus (HBV) was undertaken using 39 mammalian hepadnaviruses for which complete genome sequences were available, including representatives of all six human genotypes, as well as a large sample of small S gene sequences. Phylogenetic trees of these data were ambiguous, supporting no single place of origin for HBV, and depended heavily on the underlying model of DNA substitution. In some instances genotype F, predominant in the Americas, was the first to diverge, suggesting that the virus arose in the New World. In other trees, however, sequences from genotype B, prevalent in East Asia, were the most divergent. An attempt was also made to determine the rate of nucleotide substitution in the C open reading frame and then to date the origin of HBV. However, no relationship between time and number of substitutions was found in two independent data sets, indicating that a reliable molecular clock does not exist for these data. Both the pattern and the rate of nucleotide substitution are therefore complex phenomena in HBV and hinder any attempt to reconstruct the past spread of this virus. Received: 5 December 1998 / Accepted: 23 February 1999     

Structure and Origin of a Novel Dimeric Retroposon B1-dID

Here we describe a new short retroposon family of rodents. Like the primate Alu element consisting of two similar monomers, it is dimeric, but the left and right monomers are different and descend from B1 and ID short retroposons, respectively. Such elements (B1-dID) were found in the genomes of Gliridae, Sciuridae, Castoridae, Caviidae, and Hystricidae. Nucleotide sequences of this retroposon can be assigned to several structural variants. Phylogenetic analysis of B1-dID and related sequences suggests a possible scenario of B1-dID evolution in the context of rodent evolution. Received: 30 August 1999 / Accepted: 20 March 2000     

Coordinated Amino Acid Changes in the Evolution of Mammalian Defensins

The mammalian defensin molecule is a short, highly cationic peptide cytotoxic to both microbial and mammalian cells which is cleaved from a precursor including a signal peptide and a highly anionic propiece. A phylogenetic analysis of 28 complete sequences from five mammalian species (mouse, rat, guinea pig, rabbit, and human) showed species-specific clusters of sequences, indicating that the genes duplicated after divergence of these species. Comparison of rates of synonymous and nonsynonymous nucleotide substitution suggested that gene duplication has often been followed by a period in which diversification of the mature defensins at the amino acid level has been selectively favored. In some comparisons, it appeared that amino acid differences in this region have appeared in a nonrandom fashion so as to change the pattern of residue charges. Because it has been hypothesized that the negative charge in the propiece serves to balance the positive charge in the mature defensin and thus to prevent cytotoxicity prior to cleavage, we used a maximum likelihood method of reconstructing ancestral states in order to test whether this balance has been maintained over evolutionary time in spite of rapid diversification of the mature defensin at the amino acid level. Reconstructed ancestral sequences always maintained a charge balance between mature defensin and propiece, and changes in the net positive charge of the mature defensin were balanced by corresponding changes in the propiece. The results support the hypothesis that, in the evolution of these proteins, amino acid changes have occurred in a coordinated fashion so as to preserve an adaptive phenotype. Received: 23 October 1996 / Accepted: 7 January 1997     

Rates of Conservative and Radical Nonsynonymous Nucleotide Substitutions in Mammalian Nuclear Genes

To understand the process and mechanism of protein evolution, it is important to know what types of amino acid substitutions are more likely to be under selection and what types are mostly neutral. An amino acid substitution can be classified as either conservative or radical, depending on whether it involves a change in a certain physicochemical property of the amino acid. Assuming Kimura's two-parameter model of nucleotide substitution, I present a method for computing the numbers of conservative and radical nonsynonymous (amino acid altering) nucleotide substitutions per site and estimate these rates for 47 nuclear genes from mammals. The results are as follows. (1) The average radical/conservative rate ratio is 0.81 for charge changes, 0.85 for polarity changes, and 0.49 when both polarity and volume changes are considered. (2) The radical/conservative rate ratio is positively correlated with the nonsynonymous/synonymous rate ratio for charge changes or when both polarity and volume changes are considered. (3) Both the conservative/synonymous rate ratio and the radical/synonymous rate ratio are lower in the rodent lineage than in the primate or artiodactyl lineage, suggesting more intense purifying selection in the rodent lineage, for both conservative and radical nonsynonymous substitutions. (4) Neglecting transition/transversion bias would cause an underestimation of both radical and conservative rates and the ratio thereof. (5) Transversions induce more dramatic genetic alternations than transitions in that transversions produce more amino acid altering changes and among which, more radical changes. Received: 6 April 1999 / Accepted: 16 August 1999     

Differential Asparagine-Linked Glycosylation of Voltage-Gated K+ Channels in Mammalian Brain and in Transfected Cells

Glycosylation of ion channel proteins dramatically impacts channel function. Here we characterize the asparagine (N)-linked glycosylation of voltage-gated K⁺ channel α subunits in rat brain and transfected cells. We find that in brain Kv1.1, Kv1.2 and Kv1.4, which have a single consensus glycosylation site in the first extracellular interhelical domain, are N-glycosylated with sialic acid-rich oligosaccharide chains. Kv2.1, which has a consensus site in the second extracellular interhelical domain, is not N-glycosylated. This pattern of glycosylation is consistent between brain and transfected cells, providing compelling support for recent models relating oligosaccharide addition to the location of sites on polytopic membrane proteins. The extent of processing of N-linked chains on Kv1.1 and Kv1.2 but not Kv1.4 channels expressed in transfected cells differs from that seen for native brain channels, reflecting the different efficiencies of transport of K⁺ channel polypeptides from the endoplasmic reticulum to the Golgi apparatus. These data show that addition of sialic acid-rich N-linked oligosaccharide chains differs among highly related K⁺ channel α subunits, and given the established role of sialic acid in modulating channel function, provide evidence for differential glycosylation contributing to diversity of K⁺ channel function in mammalian brain. Received: 17 December 1998/Accepted: 20 January 1999     

A revised evolutionary history of hepatitis B virus (HBV)

Previous studies of the evolutionary history of hepatitis B virus (HBV) have been compromised by intergenotype recombination and complex patterns of nucleotide substitution, perhaps caused by differential selection pressures. We examined the phylogenetic distribution of recombination events among human HBV genotypes and found that genotypes A plus D, and genotypes B plus C, had distinct patterns of recombination suggesting differing epidemiological relationships among them. By analyzing the nonoverlapping regions of the viral genome we found strong bootstrap support for some intergenotypic groupings, with evidence of a division between human genotypes A–E from the viruses sampled from apes and human genotype F. However, the earliest events in the divergence of HBV remain uncertain. These uncertainties could not be explained by differential selection pressures, as the ratio of nonsynonymous-to-synonymous substitutions (d _N/d _S) did not vary extensively among lineages and there is no strong evidence for positive selection across the whole tree. Finally, we provide a new estimate of the mean substitution rate in HBV, 4.2 × 10⁻⁵, which suggests that divergence of HBV in humans and apes has occurred only in the last 6000 years.