Immobilized artificial membrane chromatography: a rapid and accurate HPLC method for predicting bile salt-membrane interactions

To predict bile salt-membrane interactions physiologically, we used an immobilized artificial membrane HPLC column that contains dimyristoyl-phosphatidylcholine molecules covalently linked to silica microspheres. Using a 90% aqueous (10% acetonitrile) mobile phase, 22 species of bile salts and 4 species of fusidates were eluted. Glycine conjugates displayed higher affinity for the column at pH 5.5, eluting later than their taurine-conjugated congeners, but this order was reversed at pH 6.5 and 7.4 as glycine conjugates became fully ionized. Capacity factors decreased logarithmically as functions of increasing temperature, permitting determinations of interaction enthalpies, which ranged from -2.86 to -7.67 kcal/mol. A standard curve was developed from which the enthalpy for an uncommon bile salt could be inferred from its capacity factor at room temperature. Bile salt interaction enthalpies were substantially better correlated than hydrophobic indices by octadecylsilane-HPLC (D. M. Heuman, J. Lipid Res. 1989. 30: 719-730) with equilibrium binding to small unilamellar vesicles and literature values reflecting bile salt-membrane interactions (e.g., biliary phosphatidylcholine secretion), but not with bile salt functions that do not require phospholipid (e.g., micellar cholesterol solubility). This new application should prove valuable for evaluating membrane-active physical-chemical properties as well as therapeutic potential of novel bile salts, particularly when they are available in quantities too small for study by conventional techniques.     

Interaction of uncharged bile salt derivatives with the ileal bile salt transport system

Two series of uncharged conjugated bile salt derivatives, N-conjugates of ethanolamine and 3-amino-1,2-propanediol were studied for interaction with the ileal bile salt transport system. Evidence for interaction is threefold. 1) In everted gut sac experiments more material was removed from the mucosal compartment when ileal sacs were used. 2) These derivatives inhibited the in vitro transport of taurocholate. 3) In vivo intestinal perfusion demonstrated greater absorption from ileum than from jejunum. Number three demonstrates that such interactions are followed by transmucosal movement. Their uphill transport was less than taurocholate transport. The Na(+) requirement for cholyl-3-amino-1,2-propanediol interaction with the system was greater than for taurocholate. This observation is similar to that previously observed with taurodehydrocholate, which had a greater Na(+) requirement for transport than taurocholate. Therefore removal of the anionic charge, as well as distortion of steroid shape, increases the Na(+) requirement for substrate interaction with the transport system. These observations support our hypothesis that this interaction involves two recognition components; one includes the steroid moiety, the other a coulombic interaction between the anionic bile salt and a cationic membrane site. Additionally the membrane would have an anionic group to accomodate the Na(+). Both factors (steroidal and coulombic) operate for optimal substrate attachment. Simultaneously the system's affinity for Na(+) increases and active transport then proceeds.     

Role of bile salt hydrophobicity in hepatic microtubule-dependent bile salt secretion

Under basal conditions, bile salt secretion by the liver is not affected by microtubule disruption. However, when a bile salt load is imposed on the liver, a microtubule-dependent secretion mechanism is recruited (J. Lipid Res. 1988. 29: 144-156). We tested the hypothesis that recruitment of this microtubule-dependent mechanism is influenced by the relative hydrophobicity of the bile salts being secreted. Intact male rats were depleted of bile salts by overnight biliary diversion, pretreated with colchicine (a microtubule inhibitor) or its inactive isomer, lumicolchicine (control), and reinfused intravenously with bile salts of increasing hydrophobicity (taurodehydrocholate < tauroursodeoxycholate < taurocholate) at 200 nmol/min.100 g. After 45 min, when steady-state bile salt secretion was achieved, tracer [3H]taurocholate was administered intravenously. The colchicine-insensitive component of bulk bile salt secretion was constant at approximately 130 nmol/min.100 g, and the colchicine-sensitive component increased from approximately 0 to 35 and 60 nmol/min.100 g, respectively, with reinfusion of the more hydrophobic bile salts. Retained bile salts accumulated in the liver and serum and were detectable in urine. Peak biliary secretion of [3H]taurocholate in control animals increased linearly from 15.3 to 18.0% administered dose/min with increasing hydrophobicity of the secreted bile salts (P < 0.002). In colchicine-pretreated animals, peak secretion rates decreased linearly from 13.8 to 9.2%/min (P < 0.001), with maximal inhibition in taurocholate-reinfused animals (P < 0.01). Utilization of a microtubule-dependent secretion mechanism increases with increasing bile salt hydrophobicity. This mechanism permits more efficient hepatic secretion of bile salts, but increases the susceptibility of bile salt secretion to microtubule disruption. We postulate that microtubule-dependent insertion of bile salt transporters into the canalicular membrane underlies the enhanced bile salt secretion observed when a bile salt load is imposed upon the liver.     

Membrane lipid composition and susceptibility to bile salt damage

Erythrocyte membranes with low sphingomyelin : choline-containing phospholipid ratios haemolyse at low concentrations of the bile salt, glycocholate. Erythrocytes with higher sphingomyelin : choline-containing phospholipid ratios require progressively greater concentrations of the bile salt for lysis. Sublytic concentrations of glycocholate remove phospholipid and acetylcholinesterase from the membranes. Membranes with low sphingomyelin : choline-containing phospholipid ratios lose both particulate (microvesicles of distinct composition) and 'solubilized' material, the particulate form predominating. The proportion of particulate material falls with increase of the membrane sphingomyelin : choline-containing phospholipid ratio and those membranes of highest sphingomyelin : choline-containing phospholipid ratio lose material predominantly in 'solubilized' form. Sheep erythrocytes treated to increase their content of phosphatidylcholine (and thereby reduce their membrane sphingomyelin : choline-containing phospholipid ratio) become more susceptible to lysis by glycocholate. These observations indicate a correlation between membrane lipid composition and the perturbation of membranes with bile salt; they also point to possible features of membranes capable of surviving exposure to the high bile salt concentrations of the biliary tract.     

Ionic requirements for the active ileal bile salt transport system

A new system of surface-induced profound hypothermia for infant cardiac operations has been developed in order to overcome problems inherent in the current techniques using crushed ice, water baths, and similar methods. The hypothermic chamber consists of two parts: a lower part, containing a refrigeration unit and a blower fan capable of lowering the air temperature in the chamber to -6 degrees C, and an upper part made of Plexiglas that has a completely detachable end to allow easy access to cannulas, the anesthesia hose, and the infant. A temperature panel recorder to monitor the infant's esophageal and rectal temperatures and the ambient chamber temperature is incorporated into the unit. Following evaluation in the animal laboratory, the hypothermic chamber has been successfully used in 10 infants without any complications attributable to the technique. This method provides a rapid and uniform drop of the body temperature and even skin cooling, eliminates the possibility of contact skin lesions, saves medical and paramedical personnel time in preparation of the infant and equipment, and allows observation of the child during the cooling phase. This hypothermic chamber has facilitated infant hypothermic operations.     

Molecular mechanisms of sperm-egg interactions and egg activation

The binding of acrosome reacted mammalian sperm to the egg plasma membrane initiates a series of signaling events in the egg, termed "egg activation", which lead to the completion of meiosis II and the initiation of a mitotic cell cycle. Many of these signaling events have characteristics of classical signal transduction events in somatic cells. Currently, there are two hypotheses for how sperm-induced egg activation is initiated. In the "receptor" hypothesis, the fertilizing sperm interacts with a specific egg surface receptor, and this interaction leads to signal transduction and effector activation. In the "fusion" hypothesis it is postulated that following fusion of the sperm and egg plasma membranes a soluble sperm-derived factor enters the egg's cytoplasm and activates pathways leading to egg activation. This chapter will provide an overview of the processes of cell-cell interaction and signal transduction leading to mammalian egg activation. It will concentrate on specific molecules proposed to be involved in sperm-egg interaction, signal transduction and effector mechanisms involved in egg activation, and a discussion of sperm-associated factors that have been implicated in egg activation.     

A study of the mechanisms of the salt catalyzed carbochlorination of kaolin

Continuous weighing of kaolin during carbochlorination has been used to show the superiority of NaCl as a catalyst for increasing the reaction rate and selectivity at 625° to 650 °C, and the possibility of using different types of carbon as a reductant. Initial distribution of NaCl in the kaolin was found to be of importance. A reaction mechanism involving the formation of NaAlCl₄ and alumina solubility in this salt is used to explain the effect of reactor configuration and kaolin particle geometry.     

The sister of P-glycoprotein represents the canalicular bile salt export pump of mammalian liver

Canalicular secretion of bile salts is a vital function of the vertebrate liver, yet the molecular identity of the involved ATP-dependent carrier protein has not been elucidated. We cloned the full-length cDNA of the sister of P-glycoprotein (spgp; Mr approximately 160,000) of rat liver and demonstrated that it functions as an ATP-dependent bile salt transporter in cRNA injected Xenopus laevis oocytes and in vesicles isolated from transfected Sf9 cells. The latter demonstrated a 5-fold stimulation of ATP-dependent taurocholate transport as compared with controls. This spgp-mediated taurocholate transport was stimulated solely by ATP, was inhibited by vanadate, and exhibited saturability with increasing concentrations of taurocholate (Km approximately 5 microM). Furthermore, spgp-mediated transport rates of various bile salts followed the same order of magnitude as ATP-dependent transport in canalicular rat liver plasma membrane vesicles, i.e. taurochenodeoxycholate > tauroursodeoxycholate = taurocholate > glycocholate = cholate. Tissue distribution assessed by Northern blotting revealed predominant, if not exclusive, expression of spgp in the liver, where it was further localized to the canalicular microvilli and to subcanalicular vesicles of the hepatocytes by in situ immunofluorescence and immunogold labeling studies. These results indicate that the sister of P-glycoprotein is the major canalicular bile salt export pump of mammalian liver.     

Bile acid feeding induces cholangiocyte proliferation and secretion: evidence for bile acid-regulated ductal secretion

BACKGROUND & AIMS: We have shown that taurocholate (TC) and taurolithocholate (TLC) interact in vitro with normal cholangiocytes, increasing DNA synthesis, secretin receptor (SR) gene expression, and adenosine 3',5'-cyclic monophosphate (cAMP) synthesis. To further extend these in vitro studies, we tested the hypothesis that bile acids (BAs) directly stimulate cholangiocyte proliferation and secretion in vivo. METHODS: After feeding with TC or TLC (1% for 1-4 weeks), we assessed the following in vivo: (1) ductal proliferation by both morphometry and immunohistochemistry for proliferating cell nuclear antigen (PCNA) and measurement of [3H]thymidine incorporation; and (2) the effect of secretin on bile secretion and bicarbonate secretion in vivo. Genetic expression of H3-histone and SR and intracellular cAMP levels were measured in isolated cholangiocytes. RESULTS: After BA feeding, there was an increased number of PCNA-positive cholangiocytes and an increased number of ducts compared with control rats. [3H]Thymidine incorporation, absent in control cholangiocytes, was increased in cholangiocytes from BA-fed rats. In BA-fed rats, there was increased SR gene expression (approximately 2.5-fold) and secretin-induced cAMP levels (approximately 3.0-fold) in cholangiocytes, which was associated with de novo secretin-stimulated bile flow and bicarbonate secretion. CONCLUSIONS: These data indicate that elevated BA levels stimulate ductal secretion and cholangiocyte proliferation.     

Is intracellular pH and/or intracellular bicarbonate a determinant of bile salt independent canalicular bile formation? The subject revisited

Canalicular bile formation is a complex process that involves basolateral and apical cell membrane transport, paracellular transport and vesicular transport, all of which may be subject to regulation by pH. We review the concept that apical cell membrane bicarbonate secretion promotes bile salt independent canalicular bile formation. We show that the presence of paracellular electrolyte transport imposes a severe restriction in interpreting data from ion substitution experiments aimed at demonstrating pH or bicarbonate dependent bile formation. Furthermore, we report on experiments that all show stimulation of bile flow under three disparate experimental conditions: i) intracellular alkalinization in the absence of [HCO3-]i or associated with a decrease of [HCO3-]i, ii) intracellular alkalinization with an increase of [HCO3-]i, and iii) intracellular acidification with increase of [HCO3-]i. It is suggested that both, intracellular pH and intracellular bicarbonate may modulate canalicular bile salt independent bile formation, but it remains conjectural which mechanism is the prevailing one under a given experimental setting.     

Effect of serum components on the physico-chemical properties of cationic lipid/oligonucleotide complexes and on their interactions with cells

Two behavioral rhythm phenotypes, oviposition and locomotor activity, have been compared in the four period genotypes (per+, pers, per0, and per1) of Drosophila. Period, signal-to-noise ratio, and phase were all analyzed and the genetic penetrance of the two characters was estimated. Significant rhythmicity of both oviposition and locomotor activity was evident in all four genotypes. The entrained and free-running periods of the activity rhythms of per+, pers, and per1 were within the range reported for these flies by previous workers, and rhythmic behavior was also shown by the per0 flies. The free-running period of the oviposition rhythm varied similarly between the four genotypes and showed significant correlation with that of the locomotor activity rhythm. It is suggested that both rhythm phenotypes are determined by the period gene, and estimates of the genetic penetrance of rhythmicity in oviposition and locomotor activity, based on period and signal-to-noise ratios (SNRs) of the different strains, are consistent with this hypothesis. The phase of maximum oviposition and locomotor activity showed greater variability between the genotypes and was not significantly correlated with period, suggesting that this rhythm characteristic is independent of mutations at the period locus.     

Effect of metformin on bile salt circulation and intestinal motility in type 2 diabetes mellitus

Theophylline anhydrate microcapsules with different amounts of MA/MMA copolymer (Eudragit L) were prepared by the solvent evaporation method. Qualitative as well as quantitative investigation of the drug-polymer interaction by Fourier transform infrared (FTIR) spectroscopy with a curve fitting program was undertaken. The release mechanisms of theophylline in pH 1.2 and pH 6.8 media were also studied to elucidate the effect of drug-polymer interaction on the release characteristics of microcapsules. Direct evidence for a hydrogen bonding interaction between theophylline and Eudragit L in microcapsules was obtained. Moreover, the fraction of hydrogen bonded theophylline increased with the increase of Eudragit L. The dissolution of theophylline from microcapsules exhibited an enteric-coated release property. The drug release mechanism was found to fit the Higuchi matrix model in the simulated gastric acid condition, but drug release was much more rapid in the pH 6.8 buffer solution. The drug release rate decreased as the composition of theophylline increased, and it was proportional to the fraction of hydrogen bonded theophylline. These results suggest that the increased fraction of hydrogen bonded theophylline in microencapsulation might improve the mixing and dispersibility of theophylline in the Eudragit L matrix, thus resulting in the increase of the release rate of theophylline from microcapsules.     

Use of the Vibrio harveyi toxicity test for evaluating mixture interactions of nitrobenzene and dinitrobenzene

Trauma during wartime has been the scourge of the ages. Conventional anesthesia with ether has been available since 1846 when it was demonstrated in Boston by a dentist named William Morton. Subsequently, ether was used during the Mexican-American War in 1847, and chloroform was used during the Crimean War from 1854 to 1856. Nurse anesthetists have made substantial contributions to care of the war-injured by initiating acute airway management and resuscitation efforts and by the administration of anesthesia care for critically injured war casualties undergoing surgical procedures. They have further contributed to goodwill in war-torn areas by providing anesthesia care to many civilian children and adults living in these areas of conflict. The evolution of nurse anesthesia contributions to the treatment of traumatized war casualties is the central focus of this article.     

Helix-specific interactions induce condensation of guanosine four-stranded helices in concentrated salt solutions

Deoxyguanosine-5'-monophosphate in water self-associates into stable structures, which include liquid-crystalline hexagonal and cholesteric phases. The structural unit is a four-stranded helix, composed of stacked Hoogsteen-bonded guanosine quartets. By using the osmotic stress method, we recently measured the force between helices in KCl solutions up to 2 M. In addition to the long-range electrostatic force, a short-range hydration repulsive contribution was recognized. The hydration repulsion is exponential, and shows a decay length independent from the ionic strength of the solution. Here, we report that more concentrated KCl solutions cause condensation of the guanosine helix in a hexagonal phase with constant equilibrium separation of approximately 7 A between helix surfaces. Long-range attraction, which induces the self-assembly, and short-range repulsion, which prevents the contact between the helices, are implied. By using osmotic stress, the force needed to push helices closer from the spontaneously assumed position has been measured. The attractive force was then estimated as a difference between the net force and the repulsive contribution, revealing an exponential decay length about two times larger than that of the short-range repulsion. The agreement with the helix interaction theory introduced recently by Kornyshev and Leikin (Kornyshev, A. A., and S. Leikin, 1997. Theory of interaction between helical molecules. J. Phys. Chem. 107:3656-3674) suggests that the repulsive and attractive forces originate from helix-specific interactions.     

Cellular mechanisms for developmental toxicity of chlorpyrifos: targeting the adenylyl cyclase signaling cascade

Developmental neurotoxicity caused by chlorpyrifos exposure is generally thought to target cholinesterase but chlorpyrifos may also act on cellular intermediates, such as adenylyl cyclase, that serve global functions in the coordination of cell development. In the current study, neonatal rats were exposed to apparently subtoxic doses of chlorpyrifos (no weight loss, no mortality) either on Postnatal Days 1-4 or on Postnatal Days 11-14, and the effects on components of the adenylyl cyclase cascade were evaluated in brain regions that are enriched (forebrain) or sparse (cerebellum) in cholinergic innervation, as well as in a nonneural tissue (heart). In all three, chlorpyrifos evoked deficits in multiple components of the adenylyl cyclase cascade: expression and activity of adenylyl cyclase itself, functioning of G-proteins that link neurotransmitter and hormone receptors to cyclase activity, and expression of neurotransmitter receptors that act through this cascade. Disruption of signaling function was not restricted to transduction of cholinergic signals but rather extended to adrenergic signals as well. In most cases, the adverse effects were not evident during the immediate period of chlorpyrifos administration, but appeared after a delay of several days. These results suggest that chlorpyrifos can affect cell development by altering the activity and reactivity of the adenylyl cyclase signaling cascade, a major control point for trophic regulation of cell differentiation. The effects are not restricted to cholinergic targets, nor even to the central nervous system. Hence, disruption of cell development by chlorpyrifos is likely to be more widespread than previously thought.     

Application of capillary electrophoresis of characterizing interactions between drugs and bile salts. Part I

Capillary electrophoresis offers a new way of characterizing interactions between different bile salts and drugs. The observed interactions were characterized with modified model functions known from affinity capillary electrophoresis (ACE) an micellar electrokinetic capillary electrophoresis (MECC). The methodical background of both methods is the change of the ionic mobility of the drug caused by partition between phases and aggregation with the bile salt molecules, respectively. This phenomenon is described by two different physicochemical models. A parameter estimation was carried out in order to obtain the partition coefficients KP as well as constants for the aggregate formation KA. Furthermore, an expression about the specific molar volume of the micelles and stoichiometric coefficients can be given.