Liposomes as a model for olfactory cells: changes in membrane potential in response to various odorants

Various odorants were found to depolarize azolectin liposomes. The results obtained are as follows. (1) Changes in the membrane potential of azolectin liposomes in response to various odorants were monitored by measuring changes in the fluorescence intensity of 3,3'-dipropylthiocarbocyanine iodide [disS-C3(5)]. Ten odorants examined increased the fluorescence intensity of the liposome-dye suspensions in a dose-dependent manner, which indicates that odorants depolarize the liposomes. Concentrations of odorants that depolarized the liposomes greatly varied among the odorants. There existed a good correlation between the minimum concentrations of odorants to depolarize the liposomes and the thresholds of respective odorants in the frog or porcine olfactory responses. (2) Addition of sphingomyelin (SM) to azolectin led to a large enhancement of depolarizations by nonanol, citral, and n-amyl acetate. The results indicate that lipid composition of liposomes is one of the factors that control the sensitivity to odorants. (3) Odorants changed the membrane fluidity of the liposomes, which was monitored by changes in the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH). The membrane fluidity was changed in concentration ranges of odorants similar to those where the membrane potential changes occurred, which suggests that changes in the membrane fluidity are related to generation of the membrane potential changes. (4) Changes in the membrane potential in response to odorants were electrically measured with the planar lipid bilayer made of an azolectin-SM (2:1 w/w) mixture. It was shown that odorants (nonanol, citral, and n-amyl acetate) depolarized the planar lipid bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Response of Nitella internodal cell to chemical stimulation. A model for olfactory receptor system.

Electrical response to excitable internodal cell of Nitella was studied by applying various kinds of odorants to the cell. Changes in membrane potential and resistance during responses induced by odorants were measured intracellularly under a variety of ionic environments in the media. Results were: 1) Some odorants (coumarin, isoamylacetate, methylacetate, 1-octanol, 1-butanol, 1-propanol) produced an all-or-nothing type action potential when the concentration of odorant exceeded a certain threshold. The action potential was followed by a gradual depolarization of the potential whose amplitude depended on the odorant concentration, C. Other odorants (heptanoic acid, beta-ionon) induced gradual depolarization of the membrane potential without evoking an action potential. 2) Membrane resistance Rm changed in various ways during depolarization: some odorants led to a temporal or gradual decrease in Rm, and others caused an increase in Rm when the membrane potential was depolarized by the application of odorants. 3) Magnitude of response to odorants OR was found to be represented by the following equation: OR =(alpha + beta square root I) log (C/Cth) for C greater than or equal to Cth where alpha and beta are constants for a given odorant, I the ionic strength in the medium, and Cth the threshold concentration of the odorant. 4) Plots of olfactory threshold of human and of internodal cell of Nitella gave a straight line having slope unity. 5) Local application of odorants on the internodal cell induced impulses which transmitted from the part treated by odorants to the other portion. Physico-chemical and physiological implications of the results obtained were discussed.     

Odorant-induced hyperpolarization and suppression of cAMP-activated current in newt olfactory receptor neurons

Although many studies have reported that odorants can elicit inhibitory responses as well as excitatory responses in vertebrate olfactory receptor neurons, the cellular mechanisms that underlie this inhibition are unclear. Here we examine the inhibitory effect of odorants on newt olfactory receptor neurons using whole cell patch clamp recording. At high concentrations, odorant stimulation decreased the membrane conductance and inhibited depolarization. Various odorants (anisole, isoamyl acetate, cineole, limonene and isovaleric acid) suppressed the depolarizing current in a dose-dependent manner. Furthermore, one odorant could suppress the depolarization caused by another odorant. The depolarization caused by isoamyl acetate was inhibited by anisole in cells that were excited by isoamyl acetate but not by anisole. Odorants were able to hyperpolarize cells that were depolarized by cAMP-induced conductance. Given that this inhibitory effect of odorants can affect excitation caused by other odorants, we suggest that it might play a role in coding odorants in olfactory receptor neurons.     

Membrane fluidity changes of liposomes in response to various odorants. Complexity of membrane composition and variety of adsorption sites for odorants.

Three kinds of liposomes prepared from phosphatidylcholine (PC), azolectin, and azolectin-containing membrane proteins of the canine erythrocytes were used as models for olfactory cells. To explore properties of the adsorption sites of odorants, membrane fluidity changes in response to various odorants were measured with various fluorescence dyes which monitor the fluidity at different depths and different regions of the membranes. (a) Application of various odorants changed the membrane fluidity of azolectin liposomes. The patterns of membrane fluidity changes in response to odorants having a similar odor were similar to each other and those in response to odorants having different odors were different from each other. These results suggested that odorants having a similar odor are adsorbed on a similar site and odorants having different odors are adsorbed on different sites. (b) Such variation of the pattern was not seen in liposomes of a simple composition (PC liposome). (c) In the proteoliposomes whose composition was more complex than that of azolectin liposomes, the patterns of membrane fluidity changes varied among odorants having a similar odor. It was concluded that liposomes of complex membrane composition have the variety of adsorption sites for odorants.     

Effects of changed lipid composition on responses of liposomes to various odorants: possible mechanism of odor discrimination

In a previous paper [Nomura, T., & Kurihara, K. (1987) Biochemistry (preceding paper in this issue)], we showed that azolectin liposomes are depolarized by various odorants and there is a good correlation between the responses in the liposomes and the frog or porcine olfactory responses. In this study, we examined effects of changed lipid composition on responses of liposomes to various odorants. The membrane potential changes in response to odorants were monitored with the fluorescent dye 3,3'-dipropylthiocarbocyanine iodide [diS-C3(5)]. Egg phosphatidylcholine (PC) liposomes showed depolarizing responses to nine odorants among ten odorants tested. The magnitudes of depolarization by alcohols were similar to those in azolectin liposomes, but those by other odorants were much less than those in azolectin liposomes. Addition of sphingomyelin (SM) to PC led to an increase in the magnitude of depolarization by most odorants. Addition of phosphatidylethanolamine (PE) to PC (PE/PC = 0.25) led to depolarizing responses to four odorants among six odorants tested, and a further increase in PE content (PE/PC = 0.54) led to depolarizing responses only to two odorants. Addition of SM to the lipids of this composition of PC and PE [SM/(PC + PE) = 0.22] led to depolarizing responses to four odorants again. Liposomes made of a mixture of SM, PE, and PC exhibited depolarizing responses to four odorants tested, and addition of cholesterol to the lipids [cholesterol/(PC + PE + SM) = 0.05 and 0.11] led to depolarizing responses only to two and one odorant, respectively. Thus, changes in lipid composition of liposomes led to great changes in specificity of the responses to odorants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of functional binding sites for progesterone in rat Leydig cell plasma membrane.

Steroid hormones influence cell functions by binding to intracellular receptors and then acting within the nucleus. There is now evidence that steroids affect cell functions also via interaction with plasma membrane receptors in a number of different cell types. In this regard, progesterone appears to be one of the most active steroids. In this paper, we evaluate the effects of progesterone on rat Leydig cell functions, determining variations of ion homeostasis and testosterone production. This steroid was able to effect a depolarization of the plasma membrane that was due to an influx of sodium (Na+) from the external medium since it was absent when extracellular Na+ was iso-osmotically substituted with choline chloride or sucrose. The determination of intracellular sodium concentration ([Na+]i) with the Na+ -sensitive fluorescent dye sodium-benzofuran-isophtalate (SBFI) confirmed these observations. Progesterone did not modify Leydig cell intracellular calcium concentration ([Ca2+]i) at any dose tested. Furthermore, using a cell impermeant progesterone conjugate, we demonstrated that progesterone was able to stimulate Leydig cell steroidogenesis in a dose-dependent manner. The exclusion of calcium (Ca2+) from the extracellular medium did not modify the depolarizing action of progesterone and its steroidogenetic effect while in Na+ -free medium (sucrose supplemented) progesterone-stimulated effects were completely blunted. Finally, using fluorescence microscopy with a fluorescein isothiocyanate-coupled cell impermeant progesterone conjugate, we identified plasma membrane binding sites for progesterone in rat Leydig cells. These results suggest that rat Leydig cells possess progesterone receptors located on the plasma membrane, which when occupied achieves a plasma membrane depolarization, dependent on an influx of Na+ from the external medium, and the subsequent activation of steroidogenesis.     

Grayanotoxin, veratrine, and tetrodotoxin-sensitive sodium pathways in the Schwann cell membrane of squid nerve fibers

The actions of grayanotoxin I, veratrine, and tetrodotoxin on the membrane potential of the Schwann cell were studied in the giant nerve fiber of the squid Sepioteuthis sepioidea. Schwann cells of intact nerve fibers and Schwann cells attached to axons cut lengthwise over several millimeters were utilized. The axon membrane potential in the intact nerve fibers was also monitored. The effects of grayanotoxin I and veratrine on the membrane potential of the Schwann cell were found to be similar to those they produce on the resting membrane potential of the giant axon. Thus, grayanotoxin I (1-30 muM) and veratrine (5-50 mug-jl-1), externally applied to the intact nerve fiber or to axon-free nerve fiber sheaths, produce a Schwann cell depolarization which can be reversed by decreasing the external sodium concentration or by external application of tetrodotoxin. The magnitude of these membrane potential changes is related to the concentrations of the drugs in the external medium. These results indicate the existence of sodium pathways in the electrically unexcitable Schwann cell membrane of S. sepioidea, which can be opened up by grayanotoxin I and veratrine, and afterwards are blocked by tetrodotoxin. The sodium pathways of the Schwann cell membrane appear to be different from those of the axolemma which show a voltage-dependent conductance.     

Permeability changes in the cytoplasmic membrane of Escherichia coli K-12 early after infection with bacteriophage T1.

The nature of the bacteriophage T1-induced changes in the permeability of the cytoplasmic membrane of Escherichia coli K-12 was investigated. At 20 degrees C and with glucose as a substrate, the addition of one bacteriophage per cell induced a complete and irreversible loss of K+ ions (single-hit phenomenon). K+ loss was compensated by an uptake of Na+, Li+, or choline by the cell, depending on which of these ions was the major cation in the medium. T1 depolarized the cells and inhibited 86Rb+-K+ exchange across the cytoplasmic membrane. The loss of K+ occurred independently of the Mg2+ concentration in the medium. By contrast, at low but not at high Mg2+ concentrations, T1 caused efflux of Mg2+ which in turn caused inhibition of respiration and a decrease of delta pH.     

Role of membrane potential in the response of human T lymphocytes to phytohemagglutinin

We have analyzed the role of membrane potential on T cell activation and cell proliferation. Depolarization of T lymphocytes, by increasing the extracellular concentration of K+ during a 1-hr exposure to PHA, results in a marked inhibition of cell proliferation. In parallel, depolarization of T cells prevented the normal increase in [Ca2+]i seen after PHA binding. In depolarized cells, PHA failed to induce IL 2 secretion, but, in contrast, IL 2 receptor expression was triggered normally and the cells were subsequently responsive to exogenous IL 2. Increasing [Ca2+]i in depolarized cells with the ionophore ionomycin, or bypassing the requirement for an increase in [Ca2+]i with TPA, restored the PHA-induced proliferative response in depolarized cells. These data confirm that a membrane potential-sensitive step, namely, Ca2+ influx and the resulting change in [Ca2+]i, is triggered by PHA. The inhibitory effects of depolarization are mediated through the impairment of IL 2 secretion, but not IL 2 receptor expression. T cell proliferation can therefore be regulated by altering membrane potential, which in turn modulates the extent of the change in [Ca2+]i. This study suggests a role for transmembrane potential in the regulation of the T cell proliferative response.     

Grayanotoxin opens Na channels from inside the squid axonal membrane.

External application of alpha-dihydro-grayanotoxin II (alpha-H2-GTX II) to squid giant axon under nonperfused condition caused substantial membrane depolarization. Intracellular perfusion of the fibers retarded this depolarization appreciably. Tritium-labeled alpha-dihydro-grayanotoxin II ([3H]alpha-H2-GTX II) in the external medium can permeate through the cell membrane, but permeation of alpha-H2-GTX II does not occur either with the carrier-mediated system or through the pores of the Na channel. The finding that the most hydrophilic grayanotoxin analogue, desacyl asebotoxin VII, is effective only when applied internally, strongly suggests that the receptor for grayanotoxin does not exist on the external surface of the membrane. The linear relationship between the concentration of [3H]alpha-H2-GTX II in the external medium and the count in the effluent from the perfused axon indicates that GTX II diffuses through the cell membrane's lipid phase and reaches the site of action only approached from the internal medium.