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.     

Release of proteins from the inner surface of squid axon membrane labeled with tritiated N-ethylmaleimide

Proteins in the inner surface of the squid axon membrane were labeled by intracellular perfusion of [3H]N-ethylmaleimide (NEM), which forms covalent bonds with free sulfhydryl groups. The excitability of the axon was unaffected by the [3H]NEM perfusion. After washout of the unbound label, the perfusate was monitored for the release of labeled proteins. Labeled proteins were released from the inner membrane surface by potassium depolarization of the axon only in the presence of external calcium ions. Replacement of the fluoride ion in the perfusion medium by various anions also caused labeled protein release. The order of effectiveness was SCN- greater than Br- greater than Cl- greater than F-. The extent of labeled protein release by the various anions was correlated with their effects on axonal excitability. The significance of these results is discussed.     

Membrane Potentials in Cell-free Preparations from Guinea Pig Cerebral Cortex: Effect of Depolarizing Agents and Cyclic Nucleotides

The distribution of [3H]triphenylmethylphosphonium ion between the medium and vesicular entities was examined in a cell-free, particulate preparation from guinea pig cerebral cortex. This distribution followed the Nernst relationship with regard to the external potassium ion concentration and, in physiological media, indicated the maintenance of a mean trans-membrane potential ranging from -58 to -78 mV. The neurotoxins batrachotoxin, veratridine, and grayanotoxin I, partially depolarized the preparation. Tetrodotoxin blocked the depolarization by batrachotoxin, veratridine, and gray-anotoxin I. The depolarization by these neurotoxins was potentiated by the presence of anemone toxin II and presumably reflected the response of vesicular components of neuronal origin. An additional potassium-sensitive depolarization probably represented the response of vesicular components of glial origin with an apparent transmembrane potential of -8 to -35 mV. No correlation could be demonstrated between changes in transmembrane potential and stimulation of cyclic AMP generation by a variety of agents in this preparation.     

Qualitative measurements of cytosolic calcium ion concentration within isolated guinea pig nerve endings using entrapped arsenazo III

If arsenazo III is present during homogenization of brain this metallochromic indicator is entrapped within subsequently isolated synaptosomes. A large proportion of the entrapped indicator is released upon addition of digitonin to disrupt the synaptosomal plasma membrane. A similar proportion of [3H]sucrose is also trapped within synaptosomes if present in the homogenization medium, suggesting that homogenization causes a transient opening of the nerve ending as it is chopped off from the axon. Addition of the ionophore A23187 or depolarization of the plasma membrane by adding veratridine, gramicidin or increasing external K+ changes the absorbance of the entrapped dye, with peaks of absorbance around 600 and 650 nm, typical of the arsenazo III-Ca2+ complex. The response to veratridine is inhibited by the Ca2+-channel antagonist, verapamil, while that of A23187 is unaffected. The present method provides a sensitive technique for measurements of changes in cytosolic calcium ion concentrations within nerve endings.     

Characterization of the carrier-mediated [3H]GABA release from isolated synaptic plasma membrane vesicles

Synaptic plasma membrane (SPM) vesicles were isolated under conditions which preserve most of their biochemical properties. Therefore, they appeared particularly useful to study the cytoplasmic GABA release mechanism through its neuronal transporter without interference of the exocytotic mechanism. In this work, we utilized SPM vesicles isolated from sheep brain cortex to investigate the process of [³H]GABA release induced by ouabain, veratridine and Na⁺ substitution by other monovalent cations (K⁺, Rb⁺, Li⁺, and choline). We observed that ouabain is unable to release [³H]GABA previously accumulated in the vesicles and, in our experimental conditions, it does not act as a depolarizing agent. In contrast, synaptic plasma membrane vesicles release [³H]GABA when veratridine is present in the external medium, and this process is sensitive to extravesicular Na⁺ and it is inhibited by extravesicular Ca²⁺ (1 mM) under conditions which appear to permit its entry. However, veratridine-induced [³H]GABA release does not require membrane depolarization, since this drug does not induce any significant alteration in the membrane potential, which is determined by the magnitude of the ionic gradients artificially imposed to the vesicles. The substitution of Na⁺ by other monovalent cations promotes [³H]GABA release by altering the Na⁺ concentration gradient and the membrane potential of SPM vesicles. In the case of choline and Li⁺, we observed that the fraction of [³H]GABA released relatively to the total amount of neurotransmitter released by K⁺ or Rb⁺ is about 28% and 68%, respectively. Since the replacement of Na⁺ by K⁺, Rb⁺, and Li⁺ causes different levels of membrane depolarization, and the replacement of Na⁺ by choline causes hyperpolarization of the vesicles, these results suggest that, in parallel to the [³H]GABA release, which is directly proportional to the level of membrane depolarization, this neurotransmitter can be released by decreasing the external Na⁺, which reflects an elevation of the Na⁺ concentration gradient (inout). Like veratridine-induced release, the depolarization-induced release of [³H]GABA by SPM vesicles is inhibited by Ca²⁺, which suggests that this divalent cation interfers with the cytoplasmic GABA release mechanism.Abbreviations used ATPase adenosine triphosphatase - GABA -aminobutyric acid - Mes 2 (N-morpholino)-ethanosulfonic acid - SPM synaptic plasma membranes - membrane potential     

Effect of synaptosomal cytosolic [3H]GABA pool depletion on secretory ability of alpha-latrotoxin

alpha-Latrotoxin, a presynaptic neurotoxin from the venom of Latrodectus mactans tredecimguttatus, induces massive [3H]GABA release from rat brain synaptosomes as a result of interaction with either Ca(2+)-dependent (neurexin 1 alpha or Ca(2+)-independent (latrophilin) membrane receptor. The main aim of the study was to elucidate whether the binding of alpha-latrotoxin to different types of receptors led to [3H]GABA secretion from one pool or in each case the source of neurotransmitter differs: in the presence of Ca2+ exocytosis is induced, while in the absence of Ca(2+)--outflow by mobile membrane GABA transporter from cytoplasm. We examined the effect of the depletion of cytosolic [3H]GABA pool by competitive inhibitors of the GABA transporter (nipecotic acid and 2,4-diaminobutyric acid) on the alpha-latrotoxin-stimulated neurotransmitter release. We also compared the influence of these agents on neurosecretion, evoked by depolarization with that evoked by alpha-latrotoxin. Depolarization was stimulated by 4-aminopyridine in the Ca(2+)-containing saline and high KCl in Ca(2+)-free medium. In synaptosomes treated with nipecotic acid unstimulated [3H]GABA release was significantly augmented and high KCl-evoked Ca(2+)-independent [3H]GABA release was essentially inhibited. But under the same conditions neurosecretion stimulated by alpha-latrotoxin greatly raised with respect to the control response. The similar results were obtained with the synaptosomes treated with 2,4-diaminobutyric acid. Another way to determine which of GABA pool is the target of alpha-latrotoxin action lay in analysis of the toxin effects on the preliminary depolarized synaptosomes. alpha-Latrotoxin influence was diminished by the preceding depolarization by 4-aminopyridine in Ca2+ presence. But after the high KCl stimulation effect of alpha-latrotoxin didn't change. These data suggest that alpha-latrotoxin triggers neurotransmitter release from synaptic vesicles via exocytosis. We suppose that the type of membrane receptor does not determine the mechanism of GABA release evoked by the toxin.     

Characterization of [3H]Dopamine Uptake Sites and [3H]Cocaine Recognition Sites in Primary Cultures of Mesencephalic Neurons During In Vitro Development

[3H]Dopamine uptake and [3H]cocaine binding sites were studied in primary cultures of ventral mesencephalon from 14-day-old rat embryos. Specific binding sites for [3H]cocaine and [3H]mazindol were detected only in intact cell cultures of ventral mesencephalon, and were absent in sonicated, washed membranes prepared from these cell cultures. [3H]Cocaine was not taken up by the cells through an active transport process because [3H]cocaine binding occurred also at 4 degrees C. Moreover, the possibility of [3H]cocaine entering the cells by passive diffusion and ion trapping was also excluded because extensive washing failed to remove [3H]cocaine from the cells. [3H]Cocaine binding was reduced to 6% of control when cells were permeabilized with streptolysin O (0.2 U/ml, 5 min). Taken together, these results suggest that in cultured mesencephalic neurons, [3H]cocaine may enter the cell by passive diffusion and then be sequestered by a cytosolic compartment that is lost in the process of permeabilization or sonication and washing of membrane preparations. Permeabilization of cultured neurons failed to alter the storage of [3H]dopamine. When cells were permeabilized with streptolysin O (0.2 U/ml; 5 min) after [3H]dopamine was taken up, [3H]dopamine was retained by the cells and did not leak into the incubation medium, indicating that [3H]dopamine was stored in sites that could not pass through the perforated membranes. In contrast, [3H]dopamine uptake into already permeabilized cells was reduced by 33%, suggesting that a cytosolic protein that had leaked out may play a functional role in the uptake process. In contrast to striatal membrane preparations of adult rats, [3H]cocaine binding in intact mesencephalic cell cultures was Na+ independent.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Effects of Mechanical Stimulation on Some Electrical Properties of Axons

Rapid, short duration mechanical compression of lobster giant axons by a crystal-driven stylus produces a depolarization and an increase in membrane conductance which develop immediately with compression but take several seconds to recover. The conductance increase occurs even when the depolarization is prevented electrically. If sodium is removed from the external medium or if procaine is added to it, compression produces almost no depolarization. Small bundles of myelinated frog fibers are depolarized by rapid compression but recover very rapidly (milliseconds); "off" responses are occasionally seen. The results are discussed in terms of the mechanoelectric transducer behavior of an axon membrane.     

Characterization of the Effect of Monensin on γ-Amino-n-Butyric Acid Release from Isolated Nerve Terminals

The action of the polyether antibiotic monensin on the release of gamma-[3H]amino-n-butyric acid [( 3H]GABA) from mouse brain synaptosomes is characterized. Monensin enhances the release of this amino acid transmitter in a dose-dependent manner and does not modify the efflux of the nontransmitter amino acid alpha-[3H]aminoisobutyrate. The absence of external Ca2+ fails to prevent the stimulatory effect of monensin on [3H]GABA release. Furthermore, monensin is less effective in stimulating [3H]GABA release in the presence of Ca2+. The releasing response to monensin is absolutely dependent on external Na+. The blockade of voltage-sensitive Na+ or Ca2+ channels does not modify monensin-induced release of the transmitter. Also, the blockade of the GABA uptake pathway fails to prevent the stimulatory effect of monensin on [3H]GABA release. Although monensin markedly increases Na+ permeability in synaptosomes, these data indicate that the Ca2+-independent monensin-stimulated transmitter release is not mediated by the Na+-dependent uptake pathway. It is concluded that the entrance of Na+ through monensin molecules inserted in the presynaptic membrane might be sufficient to initiate the intraterminal molecular events underlying transmitter release.     

Angiotensin II Increases Catecholamine Release from Bovine Adrenal Medulla but Does Not Enhance That Evoked by K+ Depolarization or by Carbachol

The effect of angiotensin II on catecholamine release from bovine adrenal medulla has been investigated. In retrogradely perfused, isolated bovine adrenal glands, angiotensin II increased basal efflux of catecholamines, but the presence of angiotensin II did not increase the release of catecholamines evoked either by bolus injections of the secretagogue carbachol or by depolarization with a perfusing solution containing a raised concentration of K+. In chromaffin cells maintained in primary tissue culture, angiotensin II increased 3H release from cells preloaded with [3H]-noradrenaline but did not enhance the release evoked by carbachol or by depolarization with K+. The increase in 3H release evoked by angiotensin II from chromaffin cells in tissue culture was inhibited by its analogue antagonist Sar1,Ala8-angiotensin II (saralasin) and was entirely dependent on the presence of Ca2+ in the experimental medium. These findings suggest that, in the chromaffin cells of the bovine adrenal medulla, angiotensin II acts on specific receptors to cause a calcium-dependent catecholamine release but triggers no additional response that acts synergistically with depolarizing or nicotinic stimuli to augment catecholamine release.     

Ionic permeability of K, Na, and Cl in potassium-depolarized nerve. Dependency on pH, cooperative effects, and action of tetrodotoxin.

The passive ionic membrane conductances (gj) and permeabilities (Pj) of K, Na, and Cl of crayfish (Procambarus clarkii) medial giant axons were determined in the potassium-depolarized axon and compared with that of the resting axon. Passive ionic conductances and permeabilities were found to be potassium dependent with a major conductance transition occurring around an external K concentration of 12-15 mM (Vm = -60 to -65 mV). The results showed that K, Na, and Cl conductances increased by 6.2, 6.9, and 27-fold, respectively, when external K was elevated from 5.4 to 40 mM. Permeability measurements indicated that K changed minimally with K depolarization while Na and Cl underwent an order increase in permeability. In the resting axon (K0 = 5.4 mM, pH = 7.0) PK = 1.33 X 10(-5), PCl = 1.99 X 10(-6), PNa = 1.92 X 10(-8) while in elevated potassium (K0 = 40 mM, pH 7.0), PK = 1.9 X 10(-5), PCl = 1.2 X 10(-5), and PNa = 2.7 X 10(-7) cm/s. When membrane potential is reduced to 40 mV by changes in internal ions, the conductance changes are initially small. This suggests that resting channel conductances depend also on ion environments seen by each membrane surface in addition to membrane potential. In elevated potassium, K, Na, and Cl conductances and permeabilities were measured from pH 3.8 to 11 in 0.2 pH increments. Here a cooperative transition in membrane conductance or permeability occurs when pH is altered through the imidazole pK (approximately pH 6.3) region. This cooperative conductance transition involves changes in Na and Cl but not K permeabilities. A Hill coefficient n of near 4 was found for the cooperative conductance transition of both the Na and Cl ionic channel which could be interpreted as resulting from 4 protein molecules forming each of the Na and Cl ionic channels. Tetrodotoxin reduces the Hill coefficient n to near 2 for the Na channel but does not affect the Cl channel. In the resting or depolarized axon, crosslinking membrane amino groups with DIDS reduces Cl and Na permeability. Following potassium depolarization, buried amino groups appear to be uncovered. The data here suggest that potassium depolarization produces a membrane conformation change in these ionic permeability regulatory components. A model is proposed where membrane protein, which forms the membrane ionic channels, is oriented with an accessible amino terminal group on the axon exterior. In this model the ionizable groups on protein and phospholipid have varied associations with the different ionic channel access sites for K, Na, and Cl, and these groups exert considerable control over ion permeation through their surface potentials.     

Membrane potential changes during IgE-mediated histamine release from rat basophilic leukemia cells

The membrane potential of rat basophilic leukemia cells (RBL-2H3 cell line) has been determined by monitoring the distribution of the lipophilic [3H] tetraphenylphosphonium cation between the cells and the extracellular medium. By this method, the determined potential of these cells, passively sensitized with IgE, is -93 +/- 5 mV (mean +/- SEM, interior negative). Almost 40% of this membrane potential is rapidly collapsed upon the addition of the proton carrier, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP). It is suggested that the FCCP-sensitive fraction of the total membrane potential results from the accumulation of this cation by the mitochondria, which maintains a negative membrane potential. Thus, the resting plasma membrane potential of these cells equals -55 +/- 6 mV. During the process of immunological stimulation by antibodies directed against cell membrane bound IgE, the membrane potential decreases. Moreover, there is a correlation between the extent of degranulation of the cells and the depolarization. It is concluded that in common with other secretory systems, depolarization of the plasma membrane is involved in the stimulus-secretion coupling of the histamine secreting RBL cells.     

A glucose-activated electron transfer system in the plasma membrane stimulates the H(+)-ATPase in Penicillium cyclopium.

Hyphal cells of three fungal species of the genus Penicillium reduced the nonpermeable, external electron acceptor hexabromoiridate IV (HBI IV). In Penicillium cyclopium, the rate of HBI IV reduction by hyphal cells was drastically increased by the addition of beta-glucose. The stimulation showed high specificity for this sugar and did not require its uptake and cellular metabolism. Cell wall oxidases (e.g., glucose oxidase) did not seem to be involved in the reduction of HBI IV, as no measurable H2O2 was formed from added glucose and removal of oxygen had no effect. We propose that there is a glucose-binding component outside the plasma membrane which controls transmembrane electron fluxes in response to external glucose. Reduction of HBI IV was accompanied by rapid acidification of the cellular interior (measured by confocal pH topography). Subsequently, the outer medium was acidified of the cellular interior (measured by confocal pH topography). Subsequently, the outer medium was acidified with an e-/H+ stoichiometry of > 1. In plasma membrane vesicles containing endogenous electron donors, the membrane-residing fluoroprobe Di-8-ANEPPS reported a transient depolarization of the membrane potential triggered by the external electron acceptor. Inhibitors of ATP-dependent proton pumping enhanced the extent of this depolarization, inhibited the subsequent normalization of membrane potential, and, in whole cells, reduced the amount of redox-triggered proton extrusion. From these and other findings, it is concluded that the observed trans-plasma membrane redox process activates the H(+)-ATPase via membrane depolarization and cytosolic acidification.     

Reversible and irreversible inhibition of phosphate transport in human erythrocytes by a membrane impermeant carbodiimide

Phosphate entry into chloride-loaded human erythrocytes is inhibited by treatment of cells with the water-soluble carbodiimide 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide (EAC) in the absence of added nucleophile. EAC does not penetrate the erythrocyte membrane or lead to significant intermolecular cross-linking of membrane proteins. At neutral extracellular pH in chloride-free medium, only about 50% of transport is rapidly and irreversibly inhibited, but at alkaline pH, inhibition is more rapid and complete. Inhibition by EAC was reversible in the presence of extracellular NaCl. Modification of membrane sulfhydryl groups does not prevent inhibition of phosphate transport by EAC but almost complete protection is afforded by 4,4-dinitrostilbene-2,2-disulfonic acid, a reversible competitive inhibitor of anion transport. N-(4-Azido-2-nitrophenyl)-2-aminoethylsulfonate, a reversible noncompetitive inhibitor of anion transport did not protect against EAC inhibition of transport but prevented reversal of inhibition in saline medium. Transport inhibition by [3H]EAC did not lead to specific incorporation of radioactivity into Band 3, the anion transport protein. These results suggest that inhibition of anion transport by EAC is due to modification of a carboxylic acid residue in or near the transport site accessible from the external face of the membrane. The subsequent fate of the modified carboxyl residue appears to be sensitive to the orientation of the anion transport site.     

The "late" Ca channel in squid axons

Squid giant axons were injected with aequorin and then treated with seawater containing 50 mM Ca and 100-465 mM K+. Measurements of light production suggested a phasic entry of Ca as well as an enhanced steady-state aequorin glow. After a test K+ depolarization, the aequorin-injected axon was stimulated for 30 min in Li seawater that was Ca-free, a procedure known to reduce [Na]i to about one-half the normal concentration. Reapplication of the elevated K+ test solution now showed that the Ca entry was virtually abolished by this stimulation in Li. A subsequent stimulation of the axon in Na seawater for 30 min resulted in recovery of the response to depolarization by high K+ noted in a normal fresh axon. In axons first tested for a high K+ response and then stimulated in Na seawater for 30 min (where [Na]i increases approximately 30%), there was approximately eight fold enhancement in this response to a test polarization. Axons depolarized with 465 mM K seawater in the absence of external Ca for several minutes were still capable of producing a large phasic entry of Ca when [Ca]0 was made 50 mM, which suggests that it is Ca entry itself rather than membrane depolarization that produced inactivation. Responses to stimulation at 60 pulses/s in Na seawater containing 50 mM Ca are at best only 5% of those measured with high K solutions. The response to repetitive stimulation is not measurable if [Ca]o is made 1 mM, whereas the response to steady depolarization is scarcely affected.     

Metabolism of acetylcholine in the nervous system of Aplysia californica. IV. Studies of an identified cholinergic axon

[3H]Choline, injected directly into the major axon of the identified cholinergic neuron R2, was readily incorporated into [3H]acetylcholine. Its metabolic fate was similar to that of [3H]choline injected into the cell body of R2. Over the range injected, we found that the amounts of acetylcholine formed were proportional to the amounts injected; the synthetic capability was not exceeded even when 88 pmol of [3H]choline were injected into the axon. Newly synthesized acetylcholine moved within the axon with the kinetics expected of diffusion. We could not detect any selective orthograde or retrograde transport from the site of the injection. In contrast, as indicated by experiments with colchicine, 30% of the [3H]acetylcholine formed after intrasomatic injection was selectively exported from the cell body and transported along the axon. Most of the [3H]acetylcholine was recovered in the soluble fraction after both intra-axonal and intrasomatic injection of [3H]choline; only a small fraction was particulate. The significance of large amounts of soluble acetylcholine in R2 is uncertain, and some may occur physiologically. The concentrations of choline introduced by intraneuronal injection into both cell body and axon were, however, greater than those normally available to choline acetyltransferase in the cholinergic neuron; nevertheless, these large concentrations were efficiently converted into the transmitter. The synthetic capacity of the neuron supplied with injected choline may exceed the capacity of storage vesicles and of the axonal transport process.     

Characterization of citrate transport through the plasma membrane in a carrot mutant cell line with enhanced citrate excretion

The superior ability of citrate excretion in a carrot (Daucus carota L.) mutant cell line, namely IPG (insoluble phosphate grower) [Takita et al. (1999a) Plant Cell Physiol. 40: 489] cells has been characterized in terms of citrate transport at the plasma membrane. IPG cells released about a 20-fold increase in citrate in comparison with malate, while the concentration of malate was only 35% lower than that of citrate in the cell sap. Citrate excretion was sensitive to anion channel blockers, such as niflumic acid and anthracene-9-carboxylic acid. These results indicate that IPG cells release citrate through the plasma membrane using citrate specific anion channels. The rate of citrate release from IPG cells was not affected by the concentration of aluminum (0 and 50 micro M), soluble P(i) (0 or 2 mM) and the pH (4.5-5.6) of the medium, suggesting that anion channels would not be regulated by such external conditions. Citrate excretion correlated with the H(+) efflux, possibly from the action of H(+)-ATPase on the plasma membrane. The activity of plasma membrane H(+)-ATPase was about three times higher in IPG cells than in wild-type cells, and might be involved in the high citrate excretion ability.     

Regulation of carrier-mediated and exocytotic release of [3H]GABA in rat brain synaptosomes

In this study we investigated the role of external monovalent cations, and of intracellular Ca²⁺ concentration ([Ca²⁺]i) in polarized and depolarized rat cerebral cortex synaptosomes on the release of [³H]--aminobutyric acid (³H-GABA). We found that potassium-depolarization, in the absence of Ca²⁺, of synaptosomes loaded with³H-GABA releases 7.4±2.1% of the accumulated neurotransmitter, provided that the external medium contains Na⁺, and an additional 19.0±2.5% is released upon adding 1.0 mM CaCl₂ to the exterior. The Ca²⁺-independent release component does not occur in a choline medium and it is only 3.4±0.8% of the³H-GABA accumulated in a Li⁺ medium, but both ions support the Ca²⁺-dependent release of³H-GABA (13.4±0.6% in choline and 15.4±1.5% in Li⁺), which suggests that the exocytotic release is independent of the external monovalent cation present, whereas the carrier-mediated release specifically requires Na⁺ outside. Furthermore, previous release of the cytosolic³H-GABA due to predepolarization in the absence of Ca²⁺ does not influence the amount of³H-GABA subsequently released by exocytosis due to Ca²⁺ addition (19.1±2.5% or 19.1±1.1%, respectively). In choline or Li⁺ medium, the value of the [Ca²⁺]i is raised by Na⁺/Ca²⁺ exchange to 663±75 nM or 782±54 nM, respectively, within three minutes after adding 1.0 mM Ca²⁺, in the absence of depolarization, and parallel release experiments show no release of³H-GABA in the choline medium, but a substantial release (7.1±2.1%) of³H-GABA occurs in the Li⁺ medium without depolarization. Subsequent K⁺-depolarization shows normal Ca²⁺-dependent release of³H-GABA in the choline medium (14.1±2.0%) but only 8.6±1.1% release in the Li⁺ medium, which suggests that raising the [Ca²⁺]i by Na⁺/Ca²⁺ exchange, without depolarization, supports some exocytotic release in Li⁺, but not in choline media. The role of [Ca²⁺]i and of membrane depolarization in the release process is discussed on the basis of the results obtained and other relevant observations which suggest that both Ca²⁺ and depolarization are essential for optimal exocytotic release of GABA.Special issue dedicated to Dr. Santiago Grisolia.     

Cell suspensions from porcine olfactory mucosa. Changes in membrane potential and membrane fluidity in response to various odorants

A suspension of olfactory epithelial cells was prepared from porcine olfactory mucosa and the physiological functions of the suspension were examined. The membrane potential of the cell suspension, which was monitored by measuring the fluorescence changes of rhodamine 6G, was depolarized by an increase in the K+ concentration in the external medium. Various odorants depolarized the cell suspension in a dose-dependent fashion. The magnitude of depolarization by odorants was either unchanged or slightly increased by a reduction of the concentration of Na+, Ca2+, and Cl- in the external medium, which suggests that changes in the permeabilities of specific ions are not involved in depolarization by odorants. The application of various odorants to the cell suspension induced changes in the membrane fluidity at different sites of the membrane that were monitored with various fluorescent dyes [8-anilino-1-naphthalene sulfonate, n-(9-anthroyloxy) stearic acids, 12-(9-anthroyloxy) oleic acid, and (1,6-diphenyl-1,3,5-hexatriene)], which suggests that the odorants having different odors are adsorbed on different sites in the membrane. On the basis of these results, a possible mechanism of odor discrimination is discussed.     

Different functional aspects of the group II subfamily (Types IIA and V) and type X secretory phospholipase A(2)s in regulating arachidonic acid release and prostaglandin generation. Implications of cyclooxygenase-2 induction and phospholipid scramblase-mediated cellular membrane perturbation.

We have recently reported that members of the heparin-binding group II subfamily of secretory PLA(2)s (sPLA(2)s) (types IIA and V), when transfected into 293 cells, released [(3)H]arachidonic acid (AA) preferentially in response to interleukin-1 (IL-1) and acted as "signaling" PLA(2)s that were functionally coupled with prostaglandin biosynthesis. Here we show that these group II subfamily sPLA(2)s and the type X sPLA(2) behave in a different manner, the former being more efficiently coupled with the prostaglandin-biosynthetic pathway than the latter, in 293 transfectants. Type X sPLA(2), which bound only minimally to cell surface proteoglycans, augmented the release of both [(3)H]AA and [(3)H]oleic acid in the presence of serum but not IL-1. Both types IIA and V sPLA(2), the AA released by which was efficiently converted to prostaglandin E(2), markedly augmented IL-1-induced expression of cyclooxygenase (COX)-2 in a heparin-sensitive fashion, whereas type X sPLA(2) lacked the ability to augment COX-2 expression, thereby exhibiting the poor prostaglandin E(2)-biosynthetic response unless either of the COX isozymes was forcibly introduced into type X sPLA(2)-expressing cells. Implication of phospholipid scramblase, an enzyme responsible for the perturbation of plasma membrane asymmetry, revealed that the scramblase-transfected cells became more sensitive to types IIA and V, but not X, sPLA(2), releasing both [(3)H]AA and [(3)H]oleic acid in an IL-1-independent manner. Thus, although phospholipid scramblase-mediated alteration in plasma membrane asymmetry actually led to the increased cellular susceptibility to the group II subfamily of sPLA(2)s, several lines of evidence suggest that it does not entirely mimic their actions on cells after IL-1 signaling. Interestingly, coexpression of type IIA or V, but not X, sPLA(2) and phospholipid scramblase resulted in a marked reduction in cell growth, revealing an unexplored antiproliferative aspect of particular classes of sPLA(2).