Influence of membrane structure on the kinetics of carrier-mediated ion transport through lipid bilayers.

Charge-pulse relaxation experiments of valinomycin-mediated Rb+ transport have been carried out in order to study the influence of membrane structure on carrier kinetics. From the experiment data the rate constants of association (kR) and dissociation (kD) of the ion-carrier complex as well as the rate constants of translocation of the complex (kMS) and of the free carrier (kS) could be obtained. The composition of the planar bilayer membrane was varied in a wide range. In a first series of experiments, membranes made from glycerolmonooleate dissolved in different n-alkanes (n-decane to n-hexadecane), as well as solvent-free membranes made from the same lipid by the Montal-Mueller technique were studied. The translocation rate constants kS and kMS were found to differ by less than a factor of two in the membranes of different solvent content. Much larger changes of the rate constants were observed if the structure of the fatty acid residue was varied. For instance, an increase in the number of double bonds in the C20 fatty acid from one to four resulted in an increase of kS by a factor of seven and in an increase of kMS by a factor of twenty-four. The stability constant K = kR/kD of the ion-carrier complex as well as the translocation rate constants kS and kMS were found to depend strongly on the polar headgroup of the lipid. The incorporation of cholesterol into glycerolmonooleate membranes reduced kR, kMS and kS up to seven-fold.

Kinetic independence between red cell anion exchange and urea transport.

Urea equilibrium exchange fluxes were measured in human red cells under conditions which recruit the anion transporter into an outward-facing or an inward-facing state (with respect to the anion transport site). Regardless of these conditions, urea transport always occurred at the same rate: 41 +/- 2 mol.(kg cell solids.min)-1 with 1.5 M urea at 0 degrees C. These data suggest that the pathway on the band-3 protein which mediates anion transport is kinetically uncoupled from urea transport and is probably not involved in the transport of urea across the red cell membrane.

Electrical capacity of black lipid films and of lipid bilayers made from monolayers.

Planar bilayer membranes were formed from monolayers of a series of mono-unsaturated monoglycerides and lecithins. The hydrocarbon thickness of these membranes, as calculated from the electrical capacity, increases with the length of the fatty acid chain. The specific capacity of monoolein bilayers was found to be 0.745 muF/cm-2 which is nearly twice that of a monoolein black film made in the presence of decane, but is close to that obtained after freezing out the solvent from the black film. The hydrocarbon thickness of the bilayer, as calculated with a dielectric constant of 2.1, is considerably less than twice the length of the extended hydrocarbon chain of the monoglyceride. The specific capacity (Cm) of bilayers made from monoolein monolayers showed a negligible voltage dependence, whereas the Cm increased significantly at a voltage of 150 mV in the case of Mueller-Rudin-type monoolein films with n-decane as a solvent.

Relative contributions of the slippage and tunneling mechanisms to anion net efflux from human erythrocytes.

The rates of anion net efflux from gramicidin-treated erythrocytes in the presence of a K gradient were measured at 25 degrees C, pH 7.8, as rates of loss of Ki. The experiments served to estimate the relative contributions of two hypothetical mechanisms to Cl net efflux at low extracellular Cl concentrations. Cl, Br, and NO3 net effluxes were measured into media of different Cl, Br, or NO3 concentrations, respectively, to determine and compare the relative rates of the extracellular anion-inhibitable components. They were 48, 160, and 230 mmol/(kg Hb X min), respectively, at a membrane potential of about -90 mV. This indicates that the anion-inhibitable efflux is not due solely to the return translocation of the empty transport site ("slippage") because slippage should be independent of the chemical nature of the anion. Cl net efflux was also measured as a function of the intracellular Cl concentration into media containing either 0 or 50 mM Cl. Under both conditions, net efflux was linearly dependent on Cli between 30 and 300 mM Cli and was 0 when back-extrapolated to 0 Cli. This observation is not compatible with the slippage process, which under these conditions would have been expected to be independent of Cli above 15 mM Cli. It was concluded that slippage contributes negligibly to Cl net efflux even at low extracellular anion concentrations and that the alternative process of "tunneling"--that is, movement of the anion through the anion transporter without a conformational change in a channel-type behavior--is the major, if not the sole, mechanism underlying Cl conductance.

Asymmetry in the mechanism for anion exchange in human red blood cell membranes. Evidence for reciprocating sites that react with one transported anion at a time.

The kinetics of chloride and bromide transport were examined in intact human red blood cells and resealed ghosts. Because the influx and efflux of halide ions are almost equal (less than 0.01% difference), the stimulation of the exchange flux by external halides could be determined by measuring 36Cl or 82Br efflux. When the external halide concentration was increased by replacement of isoionic, isotonic solutions of sucrose and the nontransported anion citrate, the stimulation of the exchange flux was hyperbolic and was maximum at 20 mM halide externally. The K 1/2-out, the external concentration of chloride or bromide which stimulated the efflux to half of its maximum value, was 3 and 1 mM respectively, 15-fold smaller than K 1/2-in which we found to be about equal to the K 1/2 of halide self-exchange with nearly equal internal and external concentrations. Thus, the transport mechanism behaves asymmetrically with respect to these transported halides. Bromide flux was two-fold greater in bromide-chloride heteroexchange than in bromide-bromide self-exchange but it was still much smaller than the chloride self-exchange flux. The maximum influx and efflux of bromide in exchange for chloride were roughly eqal. Thus, since the maximum transport rates in the two directions are nearly equal, the kinetics of bromide equilibrium exchange with equal concentrations on the two sides are controlled on the inside where K 1/2 is greatest. The K 1/2-out Cl was a hyperbolic function of internal chloride concentration and was proportional to the maximum flux at each internal chloride concentration. These results are evaluated in terms of two broad categories of models. We conclude that, in contrast to other ion transport systems which have been shown to have kinetics of a sequential mechanism, anion exchange is compatible with a ping-pong mechanism in which a single site reciprocates between inside- and outside-facing orientations with asymmetric K 1/2 values.

Chloride net efflux from intact erythrocytes under slippage conditions. Evidence for a positive charge on the anion binding/transport site.

Tracer chloride and potassium net efflux from valinomycin-treated human erythrocytes were measured into media of different chloride concentrations, Clo, at 25 degrees C and pH 7.8. Net efflux was maximal [45-50 mmol (kg cell solids)-1 min-1] at Clo = 0. It decreased hyperbolically with increasing Clo to 14-16 mmol (kg cell solids)-1 min-1. Half-maximal inhibition occurred at Clo = 3 mM. In the presence of the anion exchange inhibitor DNDS, net efflux was reduced to 5 mmol (kg cell solids)-1 min-1, independent of Clo. Of the three phenomenological components of net efflux, the Clo-inhibitable (DNDS-inhibitable) component was tentatively attributed to "slippage," that is, net transport mediated by the occasional return of the empty transporter. The Clo-independent (DNDS-inhibitable) component was tentatively attributed to movement of chloride through the anion transporter without the usual conformational change of the transport site on the protein ("tunneling"). These concepts of slippage and tunneling are shown to be compatible with a model that describes the anion transporter as a specialized single-site, two-barrier channel that can undergo conformational changes between two states. Net chloride efflux when the slippage component dominated (Clo = 0.7 mM) was accelerated by a more negative (inside) membrane potential. It appears that the single anion binding-and-transport site on each transporter has one net positive charge and that is neutralized when a chloride ion is bound.

Pitfall of an internal control plasmid: response of Renilla luciferase (pRL-TK) plasmid to dihydrotestosterone and dexamethasone.

The thymidine kinase promoter-Renilla luciferase reporter plasmid (pRL-TK) is commonly used as a control for transfection efficiency in the Dual-Luciferase Reporter Assay System. While investigating hormone response elements in the promoters of the androgen-dependent, epididymis-specific EP2 gene, we found that hormone treatment affected the luciferase activity of pRL-TK-transfected cells. In African Green Monkey Kidney (CV-1) cells, cotransfected transiently with a hormone-responsive promoter-firefly luciferase reporter plasmid and with pRL-TK, Renilla luciferase activity increased in response to dihydrotestosterone (DHT) and decreased in response to dexamethasone (DEX). When a thromboxane synthase promoter Renilla luciferase plasmid (pRL-TS) was used in place of pRL-TK, Renilla luciferase activity remained constant in CV-1 cells treated with DHT but decreased in CV-1 cells treated with DEX. In transfection studies, internal control plasmid expression in response to treatment must be carefully monitored to ensure proper interpretation of normalized results.

Multiple promoter and splicing mRNA variants of the epididymis-specific gene EP2.

The EP2 gene codes for a family of androgen-dependent, epididymis-specific secretory proteins. Using probes derived from human HE2 cDNA, a chimpanzee epididymal cDNA library was screened. Five variants of chimpanzee EP2 cDNA were identified. Variant 1 (EP2A) is the chimpanzee ortholog of HE2. Variant 2 (EP2B) has an alternative 5' end. Variant 3 (EP2C) has an alternative 3' end. Two additional variants were identified by reverse transcriptase-polymerase chain reaction analysis. Variant 4 (EP2D) and variant 5 (EP2E) appear to lack an exon, resulting in a shift in the open reading frame. Presumably, the 5 variants originate from the same gene and result from alternative promoters and alternative splicing. Each of the putative proteins encoded by these variant messages has a leader sequence characteristic for a secretory protein. After removal of the leader sequence, each of these proteins is predicted to consist of 1 or 2 out of 4 possible peptide modules. Two of these modules have no recognizable homology to known proteins. The other 2 modules have a distribution of cysteine residues characteristic for beta-defensins, a family of proteins with antimicrobial activity.

Recurrent laryngeal papillomatosis. Retrospective analysis of 95 patients and review of the literature.

BACKGROUND: Laryngeal papillomatosis is a benign neoplastic disease which is associated with and probably by the Human Papilloma Virus. It can be of significant importance for affected patients because of its recurrent clinical course. A great variety of therapeutic measures have been described including surgical removal either with conventional instruments or by using the laser. The aim of this study was to compare these two methods. PATIENTS: The clinical courses of all patients (53 male, 42 female) treated at the Dept. of Otolaryngology, Head and Neck Surgery, University of Kiel since 1960 were analysed retrospectively. The two most common forms of treatment, surgical removal either conventionally or with the use of the laser, were compared. RESULTS: Laryngeal papillomatosis is a disease of all ages, but often initially diagnosed in the first and fourth decade. In 25 cases the onset was before the age of 16. Puberty had no effect on the clinical course. A malignant degeneration was observed in four cases. Although the different forms of treatment did not affect the rate of recurrence, the rate of complications such as tracheostomy and glottic webs was significantly reduced after laser surgery. Since the introduction of this new form of therapy no further tracheostomies had to be performed on these patients. CONCLUSION: The term juvenile laryngeal papillomatosis should be replaced by recurrent respiratory papillomatosis. One could then make a distinction according to the age of onset, i.e. in children below the age of 16 years and in adolescents and adults older than 15 years. The occurrence of squamous cell carcinomas in patients previously treated for papillomas underlines the need for repeated histological studies. Our studies have shown that surgical treatment remains the mainstay in the management of laryngeal papillomatosis, with laser surgical technique being superior to conventional removal.

Asymmetry of the gramicidin channel in bilayers of asymmetric lipid composition: I. Single channel conductance.

Gramacidin-doped asymmetric bilayers made by the Montal-Mueller method exhibited an asymmetric current-voltage relationship. The asymmetric conductance was shown to be the product of two components, a rectifying single-channel conductance and an asymmetric voltage dependence of the reaction which leads to the conducting channel. The single-channel conductance was asymmetric in both asymmetric bilayers made of charged lipids and asymmetric bilayers made only of neutral lipids. The single-channel asymmetry decreased with increasing ion concentration. From the comparison of the single-channel conductance in symmetric and asymmetric bilayers and the dependence of the asymmetry on the solution ion concentrations, it was concluded that (1) the rate of ion entry into the channel is dependent on the lipid composition of the membrane and is asymmetric in asymmetric bilayers; (2) the entry step is rate determining at low ion concentrations; and (3) at higher ion concentrations the rate-determining step is the translocation across the main barrier in the membrane; and this translocation appears insensitive to lipid asymmetry.

Asymmetry of the gramicidin channel in bilayers of asymmetric lipid composition: II. Voltage dependence of dimerization.

The asymmetric current-voltage relationship of gramicidin-doped asymmetric bilayers made by the Montal-Mueller technique was investigated in current relaxation experiments. It was shown that, in addition to the contribution of the asymmetric single channel conductance to the asymmetry of the steady-state current-voltage relationship, there is an asymmetric voltage dependence of the step which leads to the formation of the conducting channel. This asymmetric voltage dependence could be simulated in a model assuming a membrane-internal electrical potential drop or an equivalent potential, called asymmetry potential, which could be compensated by externally applying an offset potential. Significant asymmetry potentials were found in asymmetric bilayers made of charged lipids or only of neutral lipids. The asymmetry potential was dependent on the salt composition in the aqueous phase. The factors responsible for the asymmetry potential do not appear to be of electrostatic origin. Several lines of evidence suggest that the dimerization step which leads to the conducting ion channel may be a complex series of reactions which are influenced by one or more membrane structural properties not yet characterized, in addition to the effects of the externally applied electric field.

The external anion binding site of the human erythrocyte anion transporter: DNDS binding and competition with chloride.

The interaction between chloride and the anion transport inhibitor DNDS (4,4'-dinitro stilbene-2,2'-disulfonate) at the external anion binding site of the human erythrocyte anion transporter was examined by two techniques: a) chloride tracer flux experiments in the presence of varying concentrations of DNDS, and b) DNDS equilibrium binding experiments in the presence of varying concentrations of intracellular and extracellular chloride, Cli and Clo. DNDS inhibited competitively the Clo-stimulated chloride efflux from intact red cells at 0 degree C and pH 7.8 with an inhibitor constant of 90 nM. Under the same conditions DNDS bound reversibly to one class of binding sites on intact cells with a capacity of 8.5 X 10(5) molecules/cell. Clo competitively inhibited DNDS binding with an inhibitor constant of 6 mM. In the absence of Clo the DNDS binding constant was 84 mM. The competition between chloride and DNDS was also tested in nystatintreated cells in which Clo always equaled Cli. Under these conditions the values of the DNDS binding constant and the chloride inhibitor constant were significantly larger. All these data were in quantitative agreement with a single-site, alternating access kinetic scheme with ping-pong-type kinetics that we have previously developed for modeling chloride exchange transport. The data also served to rule out special cases of an alternative two-sited sequential-type kinetic scheme. DNDS binding experiments were also performed at 10 and 20 degree C. We found that neither the DNDS binding constant nor the Clo inhibitor constant were significantly changed compared to 0 degree C.

Na+-H+ exchange in isolated myocytes from adult rat heart.

The intracellular pH (pHi) in ventricular myocytes isolated from adult rat heart was measured in suspension using the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Steady-state pHi in bicarbonate-free media [extracellular pH (pHo) = 7.4] was 7.16 +/- 0.11 at 37 degrees C. With the use of the ammonium chloride prepulse technique, pHi was acidified, and the rate of return to resting pHi was determined. Initial rate analysis of the recovery was used to characterize the kinetics of proton net efflux via the Na+-H+ exchanger. At pHo = 7.4, proton extrusion was stimulated by extracellular sodium with a K1/2 = 58 +/- 16 mM and a maximal rate of recovery of 55 +/- 7 mmol/(1 cell H2O.min). Amiloride, which inhibited greater than 90% of the observed proton movements, was a competitive inhibitor with respect to Nao, with an inhibition constant of 3.5 microM. Proton net efflux was also inhibited by extracellular protons, with a maximal flux occurring above pHo = 8 and no net efflux occurring below pHo = 6.0. Efflux was stimulated by intracellular protons over a much narrower range (pHi = 6.6-7.1). This steep dependence indicates the involvement of at least one additional proton binding site besides the intracellular transport site, in accordance with the kinetic behavior observed in other cell systems.

Molecular cloning and characterization of EPI-1, the major protein in chimpanzee (Pan troglodytes) cauda epididymal fluid.

A 27-kDa glycoprotein comprises approximately 20% of the total protein in chimpanzee (Pan troglodytes) cauda epididymal fluid. Polyclonal antibodies generated against this glycoprotein react with 27- and 25-kDa components in chimpanzee cauda epididymal fluid and in human, gorilla, chimpanzee, and monkey seminal fluid. According to microsequencing, the 27- and 25-kDa components (chimpanzee EPI-1) are identical to the cloned putative human epididymal protein HE1. Screening of a chimpanzee epididymal cDNA library enabled isolation of a cDNA clone of chimpanzee EPI-1. On the cDNA level, chimpanzee EPI-1 and human HE1 are 99% identical. Northern analysis localized chimpanzee EPI-1 mRNA to the distal caput epididymidis. With EPI-1 primers, polymerase chain reaction of reverse-transcribed rhesus monkey (Macaca mulatta) epididymal RNA enabled isolation of a rhesus monkey EPI-1 cDNA clone. The derived amino acid sequence of rhesus monkey EPI-1 is identical to chimpanzee EPI-1 and to human HE1. Northern analysis localized rhesus monkey EPI-1 mRNA to the distal caput and the proximal corpus epididymidis. Northern analysis also showed that chimpanzee EPI-1 and rhesus monkey EPI-1 gene products are expressed specifically in the epididymis and not in any other tissue examined.

Alpha 1-adrenergic stimulation of Na-H exchange in cardiac myocytes.

The activation of Na-H exchange in adult rat heart myocytes was characterized in response to a phorbol ester (phorbol 12-myristate 13-acetate) and an alpha 1-adrenergic agonist [6-fluoronorepinephrine (6F-NE)]. Transport activation was assessed by determining the initial rate with which intracellular pH (pHi) was returned from an acid pulse and by following changes in steady-state pHi; pHi was determined by a pH-sensitive fluorescent dye. Both agonists shifted the intracellular pH dependence of Na-H exchange by 0.10-0.15 pH units in the alkaline direction. This shift was prevented by the presence of sphingosine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), inhibitors of protein kinase C. The agonists also alkalinized pHi at steady state. The alkalinization by 6F-NE was blocked by prazosin and H-7. This indicates that the adrenergic stimulation of cardiac Na-H exchange is mediated by an alpha 1-adrenergic mechanism and very likely involves the activation of protein kinase C.