Biomedical subjects

S A Simon

Publications and source records attributed to S A Simon.

At least 19 recordsLinked to original sources

PubMed1998

The influence of removing extracellular Ca2+ in the desensitization responses to capsaicin, zingerone and olvanil in rat trigeminal ganglion neurons.

Desensitization is a process that describes the diminishing effect of a drug upon repeated applications. In regard to capsaicin, the pungent compound in hot pepper, it is well established that removal of extracellular calcium markedly diminishes desensitization. To explore whether this behavior extends to other analogues of capsaicin, we have determined the effect of removing extracellular calcium with capsaicin analogues, zingerone and olvanil, by whole-cell patch clamping cultured rat trigeminal ganglion neurons. Zingerone, like capsaicin, is pungent but has a shorter acyl chain, whereas olvanil is non-pungent and has a longer acyl chain. The currents evoked by 30-s applications of 30 mM zingerone or 1 microM olvanil repeated every 3 min differ in two important ways from the responses evoked by 1 microM capsaicin under these same conditions. In the presence of extracellular calcium, repeated applications of zingerone and olvanil produce nearly complete desensitization. Also in contrast to capsaicin, removing extracellular calcium for these two agonists does not diminish desensitization. These data analyses suggest the existence of calcium-independent pathways that can result in desensitization, and that pungency is not related to the phenomenon of desensitization.

Animals↗

PubMed1998

Neuronal nicotinic acetylcholine receptors in rat trigeminal ganglia.

The application of nicotine to the various epithelia served by the trigeminal nerve produces irritation and/or pain by activating neuronal nicotinic acetylcholine receptors (NnAChRS) in sensory neurons. In this study the NnAChRs were identified in rat trigeminal ganglia (TG) using RT-PCR and immunocytochemistry. With RT-PCR the subunits of NnAChRs in rat TG were determined, and with immunocytochemistry the localization of three prominent subunits (alpha 7, alpha 4 and beta 2) were localized in intact TG neurons. The relative abundance of the alpha and beta subunits were: alpha 7 approximately alpha 3 > alpha 6 > alpha 4 approximately alpha 5 > alpha 9 > or = alpha 2, and beta 2 approximately beta 3 > beta 4. This is the first report of the alpha 9 subunit in TG. Immunohistochemical studies revealed that almost all TG neurons contained alpha 7-LI and alpha 4-LI, and that 85% had beta 2-LI. For these three subunits much of the label was internalized. Immunocytochemical studies using antibodies raised against chick alpha 8 subunits did not specifically label rat TG. These data reveal that rat TG neurons contain the entire spectrum of mammalian NnAChR subunits.

Animals↗

PubMed1998

Modulation of the binding of signal peptides to lipid bilayers by dipoles near the hydrocarbon-water interface.

Interactions between signal (leader) sequences and membranes are critical to protein insertion and translocation across membranes. In this paper, circular dichroism, tryptophan fluorescence, electrophoretic mobility, dipole potential, and binding measurements were used to study the interaction of the signal sequence of the Escherichia coli LamB protein with various lipid bilayers. By modifying specific chemicophysical properties of both the signal sequence and bilayer, we analyzed some of the key factors underlying peptide-lipid interactions. We synthesized three analogues of the LamB signal peptide differing in their net charge (-2 to +4) and studied their binding to bilayers containing combinations of neutral lipids [egg phosphatidylcholine (EPC), sphingomyelin, cholesterol, ketocholesterol, and nitroxide-containing phospholipid] and a charged lipid (phosphatidylserine). All three peptides bound to EPC bilayers and underwent a random coil to alpha-helix transition upon binding. Microelectrophoresis experiments revealed that both the N and C termini were near the outer surface of the bilayer, suggesting that the peptides adopted a "hammock" configuration with both termini exposed to the aqueous phase and the core of the alpha-helix located near the hydrocarbon-water interface. The binding of these LamB peptides was not markedly dependent on the bilayer area per molecule, compressibility modulus, or dipole potential, but did depend on the charge of the peptide and bilayer interfacial region. Moreover, the binding of LamB peptides was essentially eliminated in bilayers composed of phospholipids with a nitroxide moiety at the 7 position in one of their acyl chains or in EPC bilayers containing equimolar ketocholestanol. We propose that the incorporation of nitroxide or ketone groups into the hydrocarbon region near the lipid headgroup increases the effective width of the hydrophilic interfacial region and prevents some of the hydrophobic amino acids in the alpha-helix from reaching the nonpolar hydrocarbon core, thereby diminishing the free energy of partitioning and inhibiting peptide binding. These results point to an important role for interfacial dipoles in peptide-lipid interactions.

Amino Acid Sequence↗

PubMed1998

A non-pungent resiniferatoxin analogue, phorbol 12-phenylacetate 13 acetate 20-homovanillate, reveals vanilloid receptor subtypes on rat trigeminal ganglion neurons.

Capsaicin, the vanilloid responsible for the pungent taste of hot peppers, binds to receptors found primarily in polymodal nociceptors. Capsaicin initially stimulates polymodal nociceptors and subsequently inhibits them from responding to a variety of stimuli. This property makes it useful clinically as an analgesic and anti-inflammatory compound. There is mounting, albeit indirect, evidence for the existence of several subtypes of vanilloid receptors. One such piece of evidence comes from studying analogues of capsaicin, such as phorbol 12-phenylacetate 13 acetate 20-homovanillate. This compound binds to (capsaicin) vanilloid receptors on sensory neurons, but unlike capsaicin it is non-pungent and does not produce hypothermia. To determine how sensory neurons respond to phorbol 12-phenylacetate 13 acetate 20-homovanillate, and to compare these responses with those evoked by capsaicin, whole-cell patch-clamp measurements were performed on cultured rat trigeminal ganglion neurons. It was found that 63% of the neurons held at -60 mV were activated by 3 microM, phorbol 12-phenylacetate 13 acetate 20-homovanillate, and 87% of these were also activated by 1 microM capsaicin. In a given neuron, phorbol 12-phenylacetate 13 acetate 20-homovanillate, like capsaicin, could activate kinetically distinct inward currents. The current-voltage curves characterizing phorbol 12-phenylacetate 13 acetate 20-homovanillate responses were asymmetric and had reversal potentials at -5.8 +/- 6.0 mV and 10.4 +/- 4 mV. The averaged dose-response curves for phorbol 12-phenylacetate 13 acetate 20-homovanillate were fit to the Hill equation and had binding constants (K(1/2)s) of 2.73 microM and 0.96 microM and Hill coefficients (ns) of approximately 1 for a rapidly- and slowly-activating current, respectively. These parameters are consistent with those obtained from binding experiments and calcium-influx experiments on sensory nerves. Repeated applications of phorbol 12-phenylacetate 13 acetate 20-homovanillate every 3 min caused a complete reduction in the rapidly-activating currents leaving only a reduced slowly-activating current. This provides strong evidence for the independence of these currents and the existence of subtypes of vanilloid receptors. Additional evidence for the existence of receptor subtypes is that 10 microM capsazepine, a specific and competitive inhibitor of capsaicin-evoked responses, did not inhibit the phorbol 12-phenylacetate 13 acetate 20-homovanillate-induced currents in some neurons and partially inhibited them in other neurons. Thus, there are capsazepine-sensitive and capsazepine-insensitive subtypes of vanilloid receptors. In summary, we have obtained electrophysiological and pharmacological evidence for distinct subtypes of vanilloid receptors.

Animals↗

PubMed1998

Responses of cultured rat trigeminal ganglion neurons to bitter tastants.

The initial steps in taste and olfaction result from the activation by chemical stimuli of taste receptor cells (TRCs) and olfactory receptor neurons (ORNs). In parallel with these two pathways is the chemosensitive trigeminal pathway whose neurons terminate in the oral and nasal cavities and which are activated by many of the same chemical stimuli that activate TRCs and ORNs. In a recent single unit study we investigated the responses of rat chorda tympani and glossopharnygeal neurons to a variety of bitter-tasting alkaloids, including nicotine, yohimbine, quinine, strychnine and caffeine, as well as capsaicin, the pungent ingredient in hot pepper. Here we apply many of these same compounds to cultured rat trigeminal ganglion (TG) neurons and measure changes in intracellular calcium [Ca2+]i to determine whether TG neurons will respond to these same compounds. Of the 89 neurons tested, 34% responded to 1 mM nicotine, 7% to 1 mM caffeine, 5% to 1 mM denatonium benzoate, 22% to 1 mM quinine hydrochloride, 18% to 1 mM strychnine and 55% to 1 microM capsaicin. These data suggest that neurons from the TG respond to the same bitter-tasting chemical stimuli as do TRCs and are likely to contribute information sent to the higher CNS regarding the perception of bitter/irritating chemical stimuli.

Animals↗

PubMed1997

The responses of rat trigeminal ganglion neurons to capsaicin and two nonpungent vanilloid receptor agonists, olvanil and glyceryl nonamide.

Capsaicin, the pungent ingredient in hot pepper, activates and subsequently desensitizes a subset of polymodal nociceptors. Because its initial application to skin produces pain, nonpungent analogs such as olvanil and glyceryl nonivamide (GLNVA) were synthesized to enhance its clinical use. To explore how these nonpungent analogs differ from capsaicin, whole-cell patch-clamp recordings were performed on cultured rat trigeminal ganglion neurons. In neurons held at -60 mV, capsaicin, olvanil, and GLNVA were found to activate one or two kinetically distinct inward currents. Two inward currents were also activated when extracellular Ca2+ was replaced with Ba2+ and also when intracellular chloride was replaced by aspartate. The reversal potentials of the rapidly and slowly activating currents were 15.3 +/- 6 and -4.0 +/- 2.5 mV, respectively. These data provide strong evidence for subtypes of vanilloid receptors. One difference among these agonists is that, on average, the activation kinetics of the currents evoked by 1 microM olvanil and 30 microM GLNVA are considerably slower than those evoked by 1 microM capsaicin. Measurements of the peak current, Ip, versus agonist concentration were fit to the Hill equation to yield values of the half maximal concentrations (K1/2), and the Hill coefficients (n). For capsaicin, olvanil, and GLNVA, K1/2 = 0.68, 0.59, and 27.0 microM and n = 1.38, 1.32, and 1.24, respectively. We propose that olvanil and GLNVA are nonpungent because they activate different subtypes of receptors and/or because of their activation kinetics (compared with capsaicin) are, on average, slower than the rate they inhibit action potentials from polymodal nociceptors.

Animals↗

PubMed1997

Capsazepine, a vanilloid receptor antagonist, inhibits nicotinic acetylcholine receptors in rat trigeminal ganglia.

Vanilloid receptors are activated by capsaicin, the pungent ingredient in hot pepper. They are also specifically and competitively inhibited by capsazepine (CPZ). To determine whether CPZ is specific to vanilloid receptors, its effects were tested on the currents evoked by nicotine in rat trigeminal ganglia. We found that 10 microM CPZ, a concentration frequently used to inhibit capsaicin's physiological responses attributed to capsaicin, reversibly inhibits (40%) the magnitude of the currents activated by 100 microM nicotine. We conclude that 10 microM capsazepine can alter the effects of channels other than those activated by capsaicin, and thus caution must be used in attributing all the CPZ-sensitive physiological effects to those only produced by blocking of vanilloid receptors.

Animals↗

PubMed1997

Neural responses to bitter compounds in rats.

To determine whether the idiosyncratic distribution of transduction mechanisms for bitter tastants in rat taste receptor cells (TRCs) could be inferred from the neural activity they evoke, single neuron responses to ten bitter-tasting compounds were recorded from rat glossopharyngeal (n = 30) and chorda tympani (n = 22) neurons. Responses to several 'bitter' alkaloids were obtained: 10 mM quinine-HCl, 50 mM caffeine, and 1 mM each nicotine, yohimbine, and strychnine, plus a number of non-alkaloid bitter-tasting compounds: 0.1 M KCl, 0.01 M MgCl2, and 1 mM each phenylthiocarbamide (PTC), L-tyrosine, and denatonium benzoate. To obtain some distinctions with other stimuli NaCl (0.1 M), HCl (pH 2.0), and capsaicin (10 microM) were also tested. It was found that individual neurons in both glossopharyngeal and chorda tympani nerves differed in their relative sensitivities to the various bitter stimuli. To determine relationships among these stimuli, the differences in the evoked responses between each stimulus pair were summarized in a multi-dimensional scaling space. In these analyses neither nerve showed any obvious similarity between the placements of quinine and the other bitter stimuli. Such data suggest that first-order gustatory neurons can discriminate among the above bitter stimuli. For glossopharyngeal neurons, some similarity to quinine was found only for nicotine and denatonium, and for chorda tympani neurons, some similarity to quinine was found only for KCl and MgCl2. Of the bitter compounds tested, quinine evoked the greatest response from glossopharyngeal neurons. We propose this arises because quinine can activate TRCs by more transduction mechanisms than other bitter stimuli. The results from these studies were summarized in a qualitative model for the coding of bitter tastants where the variety of transduction mechanisms for bitters are distributed among various TRCs to account for the heterogeneous responses among the neurons.

Animals↗

PubMed1996

Localization of substance P NK-1 receptors in rat tongue.

In rat tongue, neurons containing substance P terminate in connective tissue, in taste buds, and in lingual epithelium surrounding taste buds in fungiform, foliate and circumvallate papillae. Although many functions have been attributed to these neurons, virtually nothing is known about their physiological function. As a step towards this end, immunocytochemical methods were used to identify the NK-1 receptors (SPR) in rat tongue. SPR-IR was found in the basolateral membranes of taste cells in fungiform, circumvallate and foliate papillae. SPR-IR was not found in the dorsal epithelium or in any structure that could be clearly identified as a neuron. SPR-IR was also found in von Ebner's glands in circumvallate and foliate papillae and in blood vessels in connective tissue in all three papillae. These data suggest that substance P may play a role in taste and/or in oral pain.

Amino Acid Sequence↗

PubMed1996

Capsaicin activated currents in rat dorsal root ganglion cells.

Capsaicin is a pungent-tasting compound produced by plants in the Capsium family that activates a subset of primary afferent neurons associated with pain and thermoreception. Previous studies from dorsal root ganglion (DRGs) neurons suggest that many of capsaicin's physiological responses are a consequence of its activating a cation-selective current. To further characterize the responses to capsaicin whole-cell patch-clamp measurements were performed on rat DRGs to which 0.1-10 microM capsaicin was continuously applied. The capsaicin-activated currents exhibited marked variability in their thresholds, amplitude (to 15 nA), rates of desensitization, and the number of distinct maxima in the evoked current. Similar responses were found in rat trigeminal ganglion cells. The heterogeneity in the magnitude of the currents evoked by 0.1 microM capsaicin likely reflects different types of capsaicin-sensitive neurons; a result consistent with in vitro extracellular recordings from capsaicin-sensitive sensory afferents (Seno and Dray 1993).

Animals↗

PubMed1996

Structure and interactive properties of highly fluorinated phospholipid bilayers.

Because liposomes containing fluoroalkylated phospholipids are being developed for in vivo drug delivery, the structure and interactive properties of several fluoroalkylated glycerophosphocholines (PCs) were investigated by x-ray diffraction/osmotic stress, dipole potential, and hydrophobic ion binding measurements. The lipids included PCs with highly fluorinated tails on both alkyl chains and PCs with one hydrocarbon chain and one fluoroalkylated chain. Electron density profiles showed high electron density peaks in the center of the bilayer corresponding to the fluorine atoms. The height and width of these high density peaks varied systematically, depending on the number of fluorines and their position on the alkyl chains, and on whether the bilayer was in the gel or liquid crystalline phase. Wide-angle diffraction showed that in both gel and liquid crystalline bilayers the distance between adjacent alkyl chains was greater in fluoroalkylated PCs than in analogous hydrocarbon PCs. For interbilayer separations of less than about 8 A, pressure-distance relations for fluoroalkylated PCs were similar to those previously obtained from PC bilayers with hydrocarbon chains. However, for bilayer separations greater than 8A, the total repulsive pressure depended on whether the fluoroalkylated PC was in a gel or liquid-crystalline phase. We argue that these pressure-distance relations contain contributions from both hydration and entropic repulsive pressures. Dipole potentials ranged from -680 mV for PCs with both chains fluoroalkylated to -180 mV for PCs with one chain fluoroalkylated, compared to +415 mV for egg PC. The change in dipole potential as a function of subphase concentration of tetraphenyl-boron was much larger for egg PC than for fluorinated PC monolayers, indicating that the fluorine atoms modified the binding of this hydrophobic anion. Thus, compared to conventional liposomes, liposomes made from fluoroalkylated PCs have different binding properties, which may be relevant to their use as drug carriers.

Drug Carriers↗

PubMed1996

The interaction of polyphenols with bilayers: conditions for increasing bilayer adhesion.

Because proteins and other molecules with a high polyphenol content are commonly involved in adhesion processes, we are investigating the interactions between polyphenols and biological materials. A naturally occurring polyphenol that binds a variety of proteins and lipids is tannic acid (TA), which contains five digallic acid residues covalently linked to a central D-glucose. A previous study has shown that TA increases the adhesion between apposing phosphatidylcholine (PC) bilayers and over a very narrow concentration range collapses the interbilayer fluid space from about 15 A to 5 A. To determine the chemical requirements a polyphenolic molecule must possess to increase bilayer adhesion, we have synthesized several simpler TA analogs that vary in their size, shape, and number of gallic acid and hydroxyl groups. X-ray diffraction, absorbance, binding, and differential scanning calorimetry measurements were used to investigate the interaction of these polyphenolic molecules with egg PC (EPC) and dipalmitoyl PC (DPPC) bilayers. Of these synthetic polyphenols, only penta-O-galloyl-alpha-D-glucose (PGG) was able to completely mimic the effects of TA by collapsing the interbilayer fluid space from 15 A to 5 A, decreasing the dipole potential by about 300 mV, increasing the transition enthalpy of DPPC liposomes, and inducing an interdigitated phase in DPPC. Binding studies indicated that the fluid space was reduced to 5 A at an EPC:PGG mole ratio of 5:1. We conclude that these polyphenols collapse the fluid space of PC bilayers because they 1) are amphipathic and partition into the bilayers interfacial region, 2) are long enough to span the interbilayer space, 3) contain several gallic acids distributed so that they can partition simultaneously into apposing bilayers, and 4) have sufficient gallic acid residues to interact with all lipid headgroups and cover the bilayer surface. Under these conditions we conclude that the polyphenols from interbilayer bridges. We argue that these bridges are stabilized by increased adhesion arising from an increased van der Waals interaction between apposing bilayers, electrostatic interactions between the pi electrons in the phenol ring and the -(N+CH3)3 groups on the PC headgroups, decreased hydration repulsion between bilayers, and hydrogen bonds between the H-bond-donating moieties on the polyphenols and H-bond-accepting groups in the bilayer.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1996

Capsaicin and nicotine both activate a subset of rat trigeminal ganglion neurons.

Nicotine and capsaicin produce many similar physiological responses that include pain, irritation, and vasodilation. To determine whether neuronal nicotine acetylcholine receptors (nAChR) are present on capsaicin-sensitive neurons, whole cell patch-clamp recordings were performed on rat trigeminal ganglion cells. It was found that approximately 20% of the total number of neurons tested was activated by both 100 microM nicotine and 1 nM capsaicin. Other subsets of neurons were activated by only one of these compounds, whereas a fourth subset was not activated by either compound. At -60 mV, the magnitude of the capsaicin-activated currents was about three times larger than the magnitude of the nicotine-activated currents. The current-voltage relationship of the nAChR exhibited marked rectification, such that for voltages > or = 0 mV the current was essentially zero. In contrast, the current-voltage relationship of the capsaicin-activated current was ohmic from +/- 60 mV. These data indicate the existence of subsets of capsaicin-sensitive afferent neurons.

Animals↗

PubMed1996

Capsaicin-induced currents with distinct desensitization and Ca2+ dependence in rat trigeminal ganglion cells.

1. Whole cell patch-clamp records from cultured rat trigeminal ganglion cells having soma diameters ranging from 20 to 50 microM revealed that capsaicin activated two inward currents and an outward current. At -60 mV, the inward currents could be distinguished by their different peak times, which were 4.2 +/- 3.1 and 41.4 +/- 16.4 (SD) s. 2. Cells with the smallest soma diameters had the largest current densities. 3. The more rapidly activating current had a linear current-voltage relation and a reversal potential near 0 mV. 4. The more slowly activating current is not a Ca(2+)-activated Cl- current. 5. The peak of the rapid current (Ip)-capsaicin concentration (C) relationship was characterized by Ip/Ipmax = [1 + (C/Kd)n]-1, where n = 1.2 and the dissociation constant (Kd) = 0.68 microM. 6. The rapidly activating current was heterogeneous in regards to both its rate of activation and extent of desensitization. In cells bathed in buffer containing calcium and held at -60 mV, most of the capsaicin-activated currents desensitized. Removal of extracellular Ca2+ could reduce, eliminate, or have no effect on desensitization. 7. At positive holding potentials the currents very slowly desensitized, even in the presence of Ca2+. 8. Repeated 30-s applications of 1 microM capsaicin separated by 0.5, 2.5, and 5.5 min all induced tachyphylaxis. Tachyphylaxis decreased exponentially until the current remained approximately constant. Decreasing the time between capsaicin applications increased the extent of tachyphylaxis, whereas elimination of extracellular Ca2+ markedly reduced tachyphylaxis.

Animals↗

PubMed1996

Similarities and differences in the currents activated by capsaicin, piperine, and zingerone in rat trigeminal ganglion cells.

1. Capsaicin, piperine, and zingerone are natural pungent-tasting compounds found in chili pepper, black pepper, and ginger, respectively. These structurally related compounds evoke many of the same physiological responses, but at comparable concentrations capsaicin produces complete tachyphylaxis, piperine produces partial tachyphylaxis, and zingerone can either induce or not induce tachyphylaxis. Whole cell patch-clamp studies were performed on rat trigeminal ganglion cells to determine the similarities and differences between these three pungent compounds. 2. Capsaicin (1 microM) activated a variety of inward currents having peak times ranging from 2 to 46 s that desensitized to various extents ranging from 0 to 100%. The inward currents activated by zingerone (30 mM) had peak times of approximately 2 s and all currents exhibited marked desensitization. The inward currents activated by piperine (100 microM) had peak times of approximately 25 s and all exhibited a small desensitization. 3. Piperine- and zingerone-induced currents were found only in cells that could be activated by capsaicin. 4. Capsazepine (10 microM), an established antagonist of capsaicin-induced currents, inhibited the currents evoked by piperine and zingerone, suggesting that all three compounds activate vanilloid receptors. 5. Dose-response relationships for capsaicin, piperine, and zingerone obtained at a holding potential of -60 mV had threshold and apparent dissociation constants of 0.1 and 0.68 microM, 3 and 35 microM, and 1 and 15 mM, respectively. These values were consistent with those previously obtained in behavioral studies. 6. After seven 30-s applications of 1 microM capsaicin or 100 microM piperine (in a buffer with 2 mM Ca2+), each interspersed with 2-min, 50-s washes, the peak currents were inhibited by approximately 60 and 40%, respectively. In contrast, 30 mM zingerone failed to evoke a current after six applications. After complete tachyphylaxis produced by 30 mM zingerone, 1 microM capsaicin failed to evoke a current, suggesting that these two compounds cross desensitize. 7. The similar physiological responses produced by these three compounds can be rationalized by their binding to receptors and activating currents that can all be inhibited by capsazepine. Their different physiological responses evoked by these compounds can be rationalized, in part, by their very different activation and desensitization kinetics, and perhaps by the existence of different subtypes of vanilloid receptors.

Alkaloids↗

PubMed1995

Experimental tests for protrusion and undulation pressures in phospholipid bilayers.

Theoretical treatments predict that strong entropic pressures between adjacent bilayer membranes can arise from out of plane motions caused by either thermally induced bending undulations of the entire bilayer [Harbich, W., & Helfrich, W. (1984) Chem. Phys. Lipids 36, 39-63; Evans, E. A., & Parsegian, V. A. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 7132-7136] or protrusions of individual lipid molecules from the bilayer surface [Israelachvili, J. N., & Wennerström, H. (1992) J. Phys. Chem. 96, 520-531]. To determine the relative contributions of these motions to the repulsive pressure between phospholipid bilayers, the osmotic stress/X-ray diffraction method was used to measure the range and magnitude of the total repulsive pressure, and micropipet methods were used to measure the bending moduli of phosphatidylcholine bilayers containing lysophosphatidylcholine and polyunsaturated diarachidonoylphosphatidylcholine (DAPC) bilayers. In the gel phase, incorporation of equimolar lysophosphatidylcholine into phosphatidylcholine bilayers caused the hydrocarbon chains from apposing monolayers to interdigitate, but did not appreciably change the equilibrium fluid spacing in excess buffer from its control value of 12 A. In contrast, the incorporation of equimolar lysophosphatidylcholine into liquid-crystalline phase phosphatidylcholine bilayers markedly increased the range of the repulsive pressure so that equilibrium fluid separation increased from 15 to 28 A, and also decreased the bilayer bending modulus from 5.1 x 10(-13) to 1.3 x 10(-13) erg. Liquid-crystalline DAPC bilayers had intermediate values of both equilibrium fluid separation (20 A) and bending modulus (2.8 x 10(-13) erg). Analysis of these data indicates that (1) the relative importance of entropic pressures compared to the hydration pressure depends strongly on the composition and structure of the bilayer, (2) the protrusion pressure may contribute to the total repulsive pressure at large pressures or small fluid spacings, and (3) the repulsive undulation pressure, together with the attractive van der Waals pressure, is a primary factor in determining the fluid spacing at low and/or zero applied pressures in liquid-crystalline bilayers.

Electrons↗

PubMed1995

Dye-coupling in three-dimensional histoculture of rat lingual frenulum.

A three-dimensional histoculture of wet stratified squamous epithelium of rat lingual frenulum was cultured on a liquid-air interface. The tissue retained its morphology for many days in culture. During this period the vast majority of the epithelial cells remained viable and exhibited dye (lucifer yellow) coupling in all living epithelial strata. Dye coupling was determined using two methods: the conventional intracellular injection method, and a new method--"cut-loading." In the cut-loading method, an incision is made in the epithelium in the presence of dye, and intracellular diffusion of dye throughout the epithelium was measured using confocal microscopy. The basolateral surface of the lingual frenulum also acted as a substrate for neuroblastoma cells to grow without exogenously added trophic factors. These neuroblastoma cells grow neurites that establish contacts with epithelial cells. This preparation can serve as a model for investigating interactions among epithelial cells and between nerves and epithelial cells.

Animals↗

PubMed1995

Temperature dependence of the repulsive pressure between phosphatidylcholine bilayers.

Bilayer structure and interbilayer repulsive pressure were measured from 5 to 50 degrees C by the osmotic stress/x-ray diffraction method for both gel and liquid crystalline phase lipid bilayers. For gel phase dibehenoylphosphatidylcholine (DBPC) the bilayer thickness and pressure-distance relations were nearly temperature-independent, and at full hydration the equilibrium fluid spacing increased approximately 1 A, from 10 A at 5 degrees C to 11 A at 50 degrees C. In contrast, for liquid crystalline phase egg phosphatidylcholine (EPC), the bilayer thickness, equilibrium fluid spacing, and pressure-distance relation were all markedly temperature-dependent. As the temperature was increased from 5 to 50 degrees C the EPC bilayer thickness decreased approximately 4 A, and the equilibrium fluid spacing increased from 14 to 21 A. Over this temperature range there was little change in the pressure-distance relation for fluid spacings less than approximately 10 A, but a substantial increase in the total pressure for fluid spacings greater than 10 A. These data show that for both gel and liquid crystalline bilayers there is a short-range repulsive pressure that is nearly temperature-independent, whereas for liquid crystalline bilayers there is also a longer-range pressure that increases with temperature. From analysis of the energetics of dehydration we argue that the temperature-independent short-range pressure is consistent with a hydration pressure due to polarization or electrostriction of water molecules by the phosphorylcholine moiety. For the liquid crystalline phase, the 7 A increase in equilibrium fluid spacing with increasing temperature can be predicted by an increase in the undulation pressure as a consequence of a temperature-dependent decrease in bilayer bending modulus.

Gels↗