Quantitative assessment of a circulating depolarizing factor in shock.

Sustained depolarization of cell membranes and cellular edema are known to accompany various forms of circulatory shock and probably contribute to hypovolemia and cellular dysfunction. It has been proposed that a circulating protein is responsible for these effects. In the present study we have confirmed the existence of a circulating depolarizing factor (CDF) in hemorrhagic shock, burn shock, sepsis, and cardiopulmonary bypass. Plasma samples from pigs or sheep in shock were quantitatively assayed for depolarizing activity using a microelectrode method on rat diaphragm in vitro. The depolarizing effect of CDF in vitro was similar in magnitude to that of shock in situ. We conclude that CDF can entirely account for membrane depolarization during shock. The depolarizing effect of CDF was dose-dependent and saturable; it could be reversed by rinsing the diaphragm with Ringer's or control plasma. CDF activity was detectable in plasma within 5 min after a severe scald and gradually increased over the next 25 min. Resuscitation of hemorrhaged pigs, but not burned sheep, eliminated plasma CDF activity.

Regulatory volume decrease and intracellular Ca2+ in murine neuroblastoma cells studied with fluorescent probes.

The possible role of Ca2+ as a second messenger mediating regulatory volume decrease (RVD) in osmotically swollen cells was investigated in murine neural cell lines (N1E-115 and NG108-15) by means of novel microspectrofluorimetric techniques that allow simultaneous measurement of changes in cell water volume and [Ca2+]i in single cells loaded with fura-2. [Ca2+]i was measured ratiometrically, whereas the volume change was determined at the intracellular isosbestic wavelength (358 nm). Independent volume measurements were done using calcein, a fluorescent probe insensitive to intracellular ions. When challenged with approximately 40% hyposmotic solutions, the cells expanded osmometrically and then underwent RVD. Concomitant with the volume response, there was a transient increase in [Ca2+]i, whose onset preceded RVD. For hyposmotic solutions (up to approximately -40%), [Ca2+]i increased steeply with the reciprocal of the external osmotic pressure and with the cell volume. Chelation of external and internal Ca2+, with EGTA and 1,2-bis-(o -aminophenoxy) ethane-N,N,N ',N '-tetraacetic acid (BAPTA), respectively, attenuated but did not prevent RVD. This Ca2+-independent RVD proceeded even when there was a concomitant decrease in [Ca2+]i below resting levels. Similar results were obtained in cells loaded with calcein. For cells not treated with BAPTA, restoration of external Ca2+ during the relaxation of RVD elicited by Ca2+-free hyposmotic solutions produced an increase in [Ca2+]i without affecting the rate or extent of the responses. RVD and the increase in [Ca2+]i were blocked or attenuated upon the second of two approximately 40% hyposmotic challenges applied at an interval of 30-60 min. The inactivation persisted in Ca2+-free solutions. Hence, our simultaneous measurements of intracellular Ca2+ and volume in single neuroblastoma cells directly demonstrate that an increase in intracellular Ca2+ is not necessary for triggering RVD or its inactivation. The attenuation of RVD after Ca2+ chelation could occur through secondary effects or could indicate that Ca2+ is required for optimal RVD responses.

Presynaptic calcium currents at voltage-clamped excitor and inhibitor nerve terminals of crayfish.

1. A two-electrode voltage clamp was used to record calcium currents from the excitatory and inhibitory nerve terminals that innervate the crayfish (Procambarus spp.) opener muscle. Other voltage-dependent currents were blocked with tetrodotoxin, 3,4-diaminopyridine, 4-aminopyridine and tetraethylammonium. 2. The presynaptic calcium current at both excitatory and inhibitory synapses was blocked by cadmium and omega-agatoxin IVA but was not affected by omega-conotoxin GVIA, omega-conotoxin MVIIC or nifedipine, suggesting that the calcium currents flow through P-type calcium channels. 3. Current-voltage (I-V) relations at both excitatory and inhibitory synapses are similar, with current activation near -40 mV, peak current near -10 mV and current reversal at membrane potentials greater than +25 mV. I-V relations were scaled along the current axis by partial calcium current blockade with cobalt, suggesting that series resistance and space-clamp errors were small. 4. A subset of terminals on one muscle fibre was locally superfused with a physiological saline containing barium; the rest of the preparation was superfused with a physiological saline containing calcium channel antagonists. Under such conditions the characteristics of the I-V relation were very similar to the I-V relations recorded when the entire preparation was bathed in physiological levels of calcium, suggesting that the space clamp was adequate. 5. Calcium channel activation, as determined from tail current analyses, was similar when the entire preparation was bathed in physiological levels of calcium or if terminals on one muscle fibre were locally superfused with barium. 6. During a 30 ms depolarization, calcium currents inactivated to a greater extent in inhibitory than in excitatory terminals. The inactivation was of small magnitude (< 20%) and was eliminated by intracellular injection of the calcium chelator BAPTA, suggesting that the inactivation was calcium dependent. 7. These data show that biophysical and pharmacological properties of calcium currents at crayfish neuromuscular junctions resemble those found at stellate synapses in squid.

Calcium currents, transmitter release and facilitation of release at voltage-clamped crayfish nerve terminals.

1. The presynaptic terminals at crayfish (Procambarus spp.) opener neuromuscular junctions were voltage clamped. Calcium currents were measured during (ICa) and following (tail ICa) presynaptic depolarizations; EPSPs or IPSPs were simultaneously recorded from the (postsynaptic) muscle fibre directly beneath the presynaptic impalement. 2. For short (< or = 6 ms) presynaptic depolarizations, most of the transmitter release occurred during the tail ICa. EPSP or IPSP amplitudes at the end of the 6 ms pulse (end EPSP or end IPSP) increased monotonically with the integral of the ICa ([symbol: see text]ICa). The suppression potential for transmitter release was near the apparent reversal potential for ICa. 3. When the end EPSP or end IPSP amplitude was plotted against the peak ICa elicited during a presynaptic pulse (peak ICa), large and small depolarizations which evoked the same peak ICa evoked different amounts of transmitter release. The differences in transmitter release were eliminated when end EPSP amplitude was plotted against [symbol: see text] ICa, suggesting that transmitter release during a depolarization depends only upon calcium current and not upon a subsequent voltage-dependent step. 4. The synaptic transfer function of various measurements of EPSP or IPSP amplitude vs. [symbol: see text]ICa evoked during a presynaptic depolarization was a power function having an exponent of about 3. Similar measurements of EPSP amplitude vs. [symbol: see text]tail ICa evoked following a presynaptic depolarization had an exponent of about 2. 5. Facilitation of an EPSP or IPSP was not due to increases in calcium current at the test depolarization. 6. When the conditioning depolarization was increased and the test depolarization remained constant, EPSP amplitude at the test depolarization and facilitation increased . When the conditioning depolarization remained constant and the test depolarization was increased, EPSP amplitude at the test depolarization increased, while facilitation decreased. 7. Our data suggested that transmitter release at crayfish neuromuscular junctions is a non-linear function of calcium influx, and that facilitated release utilizes intracellular calcium differently from non-facilitated release. These data contradict simple models of facilitation which combine the residual calcium hypothesis with the calcium co-operativity hypothesis of non-facilitated release.

Presynaptic calcium-activated potassium channels and calcium channels at a crayfish neuromuscular junction.

1. We used a two-microelectrode current clamp to investigate various characteristics of the Ca(2+)-activated K+ conductance [gK(Ca)] and Ca2+ conductance (gCa), and transmitter release in presynaptic terminals of excitatory neuromuscular junctions in the crayfish walking leg. 2. Voltage-activated Na+ conductances (gNa) and K+ conductances [gK(v)] were blocked with tetrodotoxin and 3,4-diaminopyridine, respectively. Under these conditions, presynaptic depolarization produced by a first (conditioning) pulse admitted Ca2+ into the presynaptic terminals and activated gK(Ca), which modulated the amplitude of the depolarization produced by a second (test) pulse. The relative amount of gK(Ca) measured at the test pulse increased with increased magnitude or duration of the conditioning pulse. 3. A brief hyperpolarization immediately after a conditioning pulse substantially reduced gK(Ca). 4. gK(Ca) activation was blocked by funnel web spider toxin (a Ca2+ channel blocker) or by injection of the presynaptic terminal region with a calcium chelator, bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA). Under current-clamp conditions, gK(Ca) was not blocked by charybdotoxin or iberiotoxin [specific gK(Ca) blockers]. 5. When gK(Ca) was blocked or reduced, the amplitude of the depolarizing afterpotential of action potentials was increased. When gK(v) was blocked or reduced, the duration of action potentials was increased. 6. Intracellular injection of BAPTA into the presynaptic terminal region eliminated evoked neurotransmitter release before test pulse modulation was affected, suggesting that the K(Ca) channel had a greater sensitivity (greater affinity or lower stoichiometry) for Ca2+ than did the transmitter release machinery. BAPTA reduced neurotransmitter release by 66-78%, but did not affect facilitation of neurotransmitter release. 7. When gNa, gK(v), and gK(Ca) were blocked, we detected a membrane depolarization produced by an increase in presynaptic gCa that was eliminated by 2 mM Cd2+ or 0 mM Ca2+.

Residual free calcium is not responsible for facilitation of neurotransmitter release.

An increase in internal free calcium ([Ca2+]i) in the presynaptic terminal is often assumed to directly produce facilitation of neurotransmitter release. Using a Ca(2+)-activated potassium conductance as a bioassay for free [Ca2+]i in the presynaptic terminal of the crayfish (Procambarus clarkii) opener neuromuscular junction, we now demonstrate that free [Ca2+]i has a decay time constant (tau) of 1-4 msec, whereas facilitation of neurotransmitter release has a decay tau of 7-43 msec. In addition, facilitation of neurotransmitter release can be markedly different at times when free [Ca2+]i values and presynaptic membrane voltages are equal. We conclude that free [Ca2+]i in the presynaptic terminal is not directly responsible for facilitation of neurotransmitter release. Our data suggest that facilitation results from bound Ca2+ or some long-lived consequence of bound Ca2+.

Effects of osmotic stress on mast cell vesicles of the beige mouse.

The large size of the vesicles of beige mouse peritoneal mast cells (4 microns in diameter) facilitated the direct observation of the individual osmotic behavior of vesicles. The vesicle diameter increased as much as 73% when intact cells were perfused with a 10 mM pH buffer solution; the swelling of the vesicle membranes exceeded that of the insoluble vesicle gel matrix, which resulted in the formation of a clear space between the optically dense gel matrix and the vesicle membrane. Hypertonic solutions shrank intact vesicles of lysed cells in a nonideal manner, suggesting a limit to the compressibility of the gel matrix. The nonideality at high osmotic strengths can be adequately explained as the consequence of an excluded volume and/or a three-dimensional gel-matrix spring. The observed osmotic activity of the vesicles implies that the great majority of the histamine known to be present is reversibly bound to the gel matrix. This binding allows vesicles to store a large quantity of transmitter without doing osmotic work. The large size of the vesicles also facilitated the measurement of the kinetics of release as a collection of individual fusion events. Capacitance measurements in beige mast cells revealed little difference in the kinetics of release in hypotonic, isotonic, and hypertonic solutions, thus eliminating certain classes of models based on the osmotic theory of exocytosis for mast cells.

Presynaptic facilitation at the crayfish neuromuscular junction. Role of calcium-activated potassium conductance.

Membrane potential was recorded intracellularly near presynaptic terminals of the excitor axon of the crayfish opener neuromuscular junction (NMJ), while transmitter release was recorded postsynaptically. This study focused on the effects of a presynaptic calcium-activated potassium conductance, gK(Ca), on the transmitter release evoked by single and paired depolarizing current pulses. Blocking gK(Ca) by adding tetraethylammonium ion (TEA; 5-20 mM) to a solution containing tetrodotoxin and aminopyridines caused the relation between presynaptic potential and transmitter release to steepen and shift to less depolarized potentials. When two depolarizing current pulses were applied at 20-ms intervals with gK(Ca) not blocked, the presynaptic voltage change to the second (test) pulse was inversely related to the amplitude of the first (conditioning) pulse. This effect of the conditioning prepulse on the response to the test pulse was eliminated by 20 mM TEA and by solutions containing 0 mM Ca2+/1 mM EGTA, suggesting that the reduction in the amplitude of the test pulse was due to activation of gK(Ca) by calcium remaining from the conditioning pulse. In the absence of TEA, facilitation of transmitter release evoked by a test pulse increased as the conditioning pulse grew from -40 to -20 mV, but then decreased with further increase in the conditioning depolarization. A similar nonmonotonic relationship between facilitation and the amplitude of the conditioning depolarization was reported in previous studies using extracellular recording, and interpreted as supporting an additional voltage-dependent step in the activation of transmitter release. We suggest that this result was due instead to activation of a gK(Ca) by the conditioning depolarization, since facilitation of transmitter release increased monotonically with the amplitude of the conditioning depolarization, and the early time course of the decay of facilitation was prolonged when gK(Ca) was blocked. The different time courses for decay of the presynaptic potential (20 ms) and facilitation (greater than 50 ms) suggest either that residual free calcium does not account for facilitation at the crayfish NMJ or that the transmitter release mechanism has a markedly higher affinity or stoichiometry for internal free calcium than does gK(Ca). Finally, our data suggest that the calcium channels responsible for transmitter release at the crayfish NMJ are not of the L, N, or T type.

Calcium-activated potassium conductance in presynaptic terminals at the crayfish neuromuscular junction.

Membrane potential changes that typically evoke transmitter release were studied by recording intracellularly from the excitor axon near presynaptic terminals of the crayfish opener neuromuscular junction. Depolarization of the presynaptic terminal with intracellular current pulses activated a conductance that caused a decrease in depolarization during the constant current pulse. This conductance was identified as a calcium-activated potassium conductance, gK(Ca), by its disappearance in a zero-calcium/EGTA medium and its block by cadmium, barium, tetraethylammonium ions, and charybdotoxin. In addition to gK(Ca), a delayed rectifier potassium conductance (gK) is present in or near the presynaptic terminal. Both these potassium conductances are involved in the repolarization of the membrane during a presynaptic action potential.

Ionic control of the size of the vesicle matrix of beige mouse mast cells.

Isolated matrices of the giant secretory vesicles of mast cells of the beige mouse were reliably produced by the osmotic lysis of isolated vesicles. These matrices maintained their form, and their sizes were easily measured using Nomarski optics. The size of the matrix depended on the ionic composition of the bathing solution. The physiologically relevant ions, histamine and serotonin, contracted the matrix. Multivalent cations condensed the matrix relative to univalents. Ag+, acid pH (below 5), and basic pH (above 9) expanded the matrix. In the presence of 10 mM histamine, lowering the pH from 9 to 5 contracted the matrix more than can be attributed to the pH-dependent matrix contraction in zero histamine. The nontitratable organic cation, dimethonium, contracts the matrix with little effect of pH in the range of 5-9. These results suggest that histamine acts as a matrix contractor in the divalent form. The dose-response (contraction) relation for histamine was gradual from micromolar to 316 mM (millimolar) histamine. Experiments with mixtures of histamine and sodium show antagonistic effects on the matrix but are inconsistent with either a model where ions compete for identical sites or a parallel model where ions interact with separate independent sites. In vigorous histamine washoff experiments, the half time for vesicle expansion in 10(-4) M pH buffer was approximately 4 s; in isotonic NaCl solution, it was 0.5 s. When 1 M histamine was presented to closely apposed matrices, fusion resulted. The matrix material returned to its initial shape after being mechanically deformed with a glass probe. These results suggest that the matrix size is controlled by its ion exchange properties. The matrix expansion can quantitatively account for the vesicular size increase observed upon exocytosis (as a postfusional event) and the osmotic nonideality of intact vesicles. The mechanical expansion is probably significant in the widening of the exocytotic pore and the dispersal of the vesicular contents.

On the time course of surgically induced compensatory muscle hypertrophication of the rat plantaris muscle.

1. The time course of the hypertrophy and hyperplasia of the rat plantaris muscle was determined from measurements of total muscle mass and cross-section analysis of fixed muscle. 2. Muscle enlargement was induced by the surgical removal of the plantaris synergist muscles, the gastrocnemius and the soleus. 3. From the date of surgery through the third post-operative week, muscle enlargement is due to fiber hypertrophication (approximately 100% increase in diameter). After post-operative week three, muscle enlargement is due to a combination of hyperplasia and hypertrophy. At week four the cross-sectional areas return to control values. 4. The neuromuscular junction area was determined by measuring Karnovsky stained post-synaptic membrane. Only a modest 10-30% increase was noted at weeks 2 and 3 with a return to control levels at week 4. The differences were not statistically different.

The interaction of intracellular Mg2+ and pH on Cl- fluxes associated with intracellular pH regulation in barnacle muscle fibers.

The intracellular dialysis technique was used to measure unidirectional Cl- fluxes and net acid extrusion by single muscle fibers from the giant barnacle. Decreasing pHi below normal levels of 7.35 stimulated both Cl- efflux and influx. These increases of Cl- fluxes were blocked by disulfonic acid stilbene derivatives such as SITS and DIDS. The SITS-sensitive Cl- efflux was sharply dependent upon pHi, increasing approximately 20-fold as pHi was decreased from 7.35 to 6.7. Under conditions of normal intracellular Mg2+ concentration, the apparent pKa for the activation of Cl- efflux was 7.0. We found that raising [Mg2+]i, but not [Mg2+]o, had a pronounced inhibitory effect on both SITS-sensitive unidirectional Cl- fluxes as well as on SITS-sensitive net acid extrusion. Increasing [Mg2+]i shifted the apparent pKa of Cl- efflux to a more acid value without affecting the maximal flux that could be attained. This relation between pHi and [Mg2+]i on SITS-sensitive Cl- efflux is consistent with a competition between H ions and Mg ions. We conclude that the SITS-inhibitable Cl- fluxes are mediated by the pHi-regulatory transport mechanism and that changes of intracellular Mg2+ levels can modify the activity of the pHi regulator/anion transporter.

Inhibition of sodium currents by local anesthetics in chloramine-T-treated squid axons. The role of channel activation.

In order to test the requirement of Na channel inactivation for the action of local anesthetics, we investigated the inhibitory effects of quaternary and tertiary amine anesthetics on normally inactivating and noninactivating Na currents in squid axons under voltage clamp. Either the enzymatic mixture pronase, or chloramine-T (CT), a noncleaving, oxidizing reagent, was used to abolish Na channel inactivation. We found that both the local anesthetics QX-314 and etidocaine, when perfused internally at 1 mM, elicited a "tonic" (resting) block of Na currents, a "time-dependent" block that increased during single depolarizations, and a "use-dependent" (phasic) block that accumulated as a result of repetitive depolarizations. All three effects occurred in both control and CT-treated axons. As in previous reports, little time-dependent or phasic block by QX-314 appeared in pronase-treated axons, although tonic block remained. Time-dependent block was greatest and fastest at large depolarizations (Em greater than +60 mV) for both the control and CT-treated axons. The recovery kinetics from phasic block were the same in control and CT-modified axons. The voltage dependence of the steady state phasic block in CT-treated axons differed from that in the controls; an 8-10% reduction of the maximum phasic block and a steepening and shift of the voltage dependence in the hyperpolarizing direction resulted from CT treatment. The results show that these anesthetics can bind rapidly to open Na channels in a voltage-dependent manner, with no requirement for fast inactivation. We propose that the rapid phasic blocking reactions in nerve are consequences primarily of channel activation, mediated by binding of anesthetics to open channels, and that the voltage dependence of phasic block arises directly from that of channel activation.

Effects of saxitoxin analogues and ligand competition on sodium currents of squid axons.

Saxitoxin (STX) and several STX analogues from dinoflagellates (genus Protogonyaulax) block sodium conductance in squid giant axons with variable potencies. Toxins, analyzed under voltage clamp, are 21-sulfosaxitoxin, 21-sulfosaxitoxin 11 alpha-hydroxysulfate, 21-sulfosaxitoxin 11 beta-hydroxysulfate, (B1, C1, C2, respectively) and gonyautoxins 2 and 3. The potency sequence for the toxins examined is STX greater than gonyautoxin 3 greater than B1 greater than C2 greater than gonyautoxin 2 much greater than C1. Guanidine, when substituted for sodium in external seawater, reduced the potency of STX to block inward current but did not affect tetrodotoxin activity. Methylguanidine also reduced the ability of STX to block outward sodium current. Inhibitory constants for guanidine and methylguanidine were 116 and 187 mM, respectively. Competition can be explained by binding at or near the toxin binding site but not by surface potential alteration.

Removal of sodium channel inactivation in squid axon by the oxidant chloramine-T.

We have investigated the effects of a mild oxidant, chloramine-T(CT), on the sodium and potassium currents of squid axons under voltage-clamp conditions. Sodium channel inactivation of squid giant axons can be completely removed by CT at neutral pH. Internal and external CT treatment are both effective. CT apparently removes inactivation in an irreversible, all-or-none manner. The activation process of sodium channels is little affected, as judged from the voltage dependence of peak sodium currents, the rising phase of sodium currents, and the time course of tail currents following the repolarization. The removal of inactivation by CT is pH-dependent; higher pH decreases the removal rate, whereas lower pH increases it. Internal metabisulfite, a strong reductant, does not protect inactivation from the action of external CT, nor does external metabisulfite protect from internal CT application. CT slightly depresses the peak potassium currents at comparable concentrations but has no apparent effects on their kinetics. Our results suggest that the neutral form of CT modifies an embedded methionine residue that is involved in sodium channel inactivation.

Intracellular pH and Na fluxes in barnacle muscle with evidence for reversal of the ionic mechanism of intracellular pH regulation.

The ion transport mechanism that regulates intracellular pH (pHi) in giant barnacle muscle fibers was studied by measuring pHi and unidirectional Na+ fluxes in internally dialyzed fibers. The overall process normally results in a net acid extrusion from the cell, presumably by a membrane transport mechanism that exchanges external Na+ and HCO-3 for internal Cl- and possibly H+. However, we found that net transport can be reversed either by lowering [HCO-3]o and pHo or by reducing [Na+]o. This reversal (acid uptake) required external Cl-, was stimulated by raising [Na+]i, and was blocked by SITS. When the transporter was operating in the net forward direction (acid extrusion), we found a unidirectional Na+ influx of approximately 60 pmol . cm-2 . s-1, which required external HCO-3 and internal Cl- and was stimulated by cyclic AMP and blocked by SITS or DIDS. These properties of the Na+ influx are all shared with the net acid extrusion process. We also found that under conditions of net forward transport, the pHi-regulating system mediated a unidirectional Na+ efflux, which was significantly smaller than the simultaneous Na+ influx. These data are consistent with a reversible transport mechanism which, even when operating in the net forward direction, mediates a small amount of reversed transport. We also found that the ouabain-sensitive Na+ efflux was sharply inhibited by acidic pHi, being totally absent at pHi values below approximately 6.8.

Chemical modification of excitable membranes.

We have presented the effects of several chemical modification procedures on the properties of excitable membranes. The approach represents a middle ground between the biochemical approach of total separation of the proteins responsible for excitability and the biophysical approach in which only the physical parameters associated with the proteins are described. The results from investigations using group specific probes have begun to provide some picture, albeit a sketchy one, of the functional architecture of the proteins associated with excitability. As such, the investigations provide important information about necessary residues and their conformational arrangement in the intact protein. This knowledge will be especially useful in understanding excitability once the primary amino acid structure of the excitability proteins is determined.

Cyclic AMP-stimulated chloride fluxes in dialyzed barnacle muscle fibers.

Unidirectional chloride efflux and influx were studied in giant barnacle muscle fibers that were internally dialyzed. When cyclic 3'5'-adenosine monophosphate (cAMP) was included in the dialysis fluid, both unidirectional fluxes were stimulated by about the same amount. This stimulation was not associated with measurable changes either in membrane electrical conductance or with net movements of chloride. The stimulation required the trans-side presence of chloride. The stimulated flux was inhibited by the sulfonic acid stilbene derivatives 4-acetamido-4'-isothiocyanostilbene-2',2'-disulfonate (SITS) and 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) or by furosemide. When cAMP was presented in high concentrations (10-5 M), the effect on chloride fluxes was characterized by a desensitization phenomenon. This desensitization was not the result of an increased amount of phosphodiesterase activity, but may be related to ATP and/or intracellular calcium levels. These results further support the hypothesis that the barnacle sarcolemma possesses a specialized chloride transport mechanism that largely engages in Cl-Cl exchange under conditions of normal intracellular pH.

Lipid vesicle-mediated alterations of membrane cholesterol levels: effects on Na+ and K+ currents in squid axon.

We demonstrate that cholesterol can exchange from sonicated lipid vesicles to a perfused squid axon membrane and that vesicles with varying cholesterol/phospholipid (C/P) mole ratios can be used to achieve either net loading or net depletion of axon membrane cholesterol. Two types of evidence were obtained which show that net loading or depletion of cholesterol was achieved: (i) changes in the cholesterol/phospholipid (C/P) mole ratios of axons, and (ii) visualization of cholesterol depleted from the preparation by cholesterol-free vesicles by thin-layer chromatography. The C/P mole ratios indicate that cholesterol levels in the preparation were increased or decreased by 30-40%. Increasing or decreasing membrane cholesterol levels were ineffective in altering the Na+ or K+ occurrents in voltage-clamped axons. In addition, we determined that cholesterol "flip-flop" across the axonal membrane occurred with a t 1/2 of 7.3 to 15.3 min.