Alteration by xylocaine (lidocaine) and its derivatives of the time course of the end plate potential

Xylocaine and its derivatives act specifically at the neuromuscular junction within the concentration range 0.05 to 2.0 mM. The charged form is the active form of the drugs. There is no correlation between "local anesthetic" activity and effect at the junction. Like d-tubocurarine, these drugs have little or no effect on quantum content, acetylcholinesterase activity, or the passive impedance of the muscle fiber. Yet they produce end plate potentials characterized by a brief, early component and a late, greatly prolonged component, as does procaine. Analysis of these changes in time course suggests that the drugs have little or no effect before receptors are activated by acetylcholine, but cause a decreased and often greatly prolonged response. Clear structure-activity relations indicate that the receptor to which the drugs bind to produce the prolonged response can be the receptor for acetylcholine. Comparison of the effects of the drugs on the end plate potential and on the response to iontophoretically applied acetylcholine also shows that the effects of Xylocaine depend on the time course of receptor activation and are quite different from the effects of d-tubocurarine.     

Effects of Ca2+ and vitamin E on posttranslational regulation of acetylcholine receptor expression in the somata of identified molluscan neurons

The effects of Ca²⁺ and vitamin E (-tocopherol) on acetylcholine (Ach)-induced Cl^– currents in LP11 and RBc4 neurons of the snail Helix pomatia have been studied. Injection of Ca²⁺ into the cells and application of vitamin E (10^–5 mole/liter) induced the appearance of potentiation of Ach-induced currents in membrane parts more remote from the axon than the Ach-sensitive regions in the control. The Hill coefficient (n) for such Ach receptors was equal to 0.8, unlike 1.8 for Ach receptors active in the control. Arachidonic acid (10^–5 mole/liter) and phorbol ester TPA (10^–6 mole/liter) inhibited Ach responses, while oleoylacetyglycerol (10^–6 mole/liter) produced no effect. Calmidazolium (10^–6 mole/liter) decreased the effects of Ca²⁺ and vitamin E on Ach responses, while nordihydroquiaretic acid (5 · 10^–6 mole/liter) enhanced the modulating effect of vitamin E and weakened that of arachidonic acid. It is suggested that the expression of Ach receptors activated by Ca²⁺ and vitamin E is mediated through posttranslational mechanisms, since cycloheximide and actinomycin D, inhibitors of protein synthesis, did not influence the effects of C²⁺ and vitamin E. The mechanisms responsible for the stimulating effects of Ca²⁺ and vitamin E are discussed.Translated from Neirofiziologiya, Vol. 25, No. 1, pp. 31–39, January–February, 1993.     

Mechanism of action of snake venom α-neurotoxins on nicotinic acetylcholine receptors of rabbit sympathetic ganglion neurons

Currents evoked by iontophoretic applications of acetylcholine and postsynaptic currents evoked by single stimulation of the cervical sympathetic nerve were recorded in neurons of the isolated rabbit superior cervical ganglion with membrane voltage clamped and muscarinic acetylcholine receptors blocked by atropine (10^?6 M). The α-neurotoxins from snake venom (α-bungarotoxin and α-cobratoxin) in a concentration of 10^?6 M caused an increase in amplitude (potentiation) of the acetylcholine current, inhibition of that current, or initial potentiation followed by inhibition, in different neurons. Spectral analysis of the fluctuations of this current showed that α-neurotoxins affect neither the current through a single channel nor the duration of the open state of long-living channels (evidently extrasynaptic), but they approximately double the duration of the open state of long-living channels. This last effect in all probability causes potentiation of the acetylcholine current. The α-neurotoxins also depressed the amplitude of the postsynaptic current evoked by sympathetic nerve stimulation (on average by 44%) and lengthened its decline (on average by 24%). It is postulated that α-neurotoxins may both block and modify activity of the receptor-channel complex in the neurons tested, lengthening the duration of its open state. This latter mechanism of action of α-neurotoxins is exhibited only in long-living channels, evidence that the phamacological properties of the two populations of channels connected with nicotinic acetylcholine receptors are not identical.     

Reversible loss of acetylcholine receptor clusters at the developing rat neuromuscular junction

Exposure of sternomastoid muscles excised from 16-day embryonic rats to medium depleted of Ca²⁺ or containing high concentrations of KCl leads to extensive loss of aggregates of acetylcholine receptors newly formed at the motor end plate region. Upon restoration of Ca²⁺ or removal of excess KCl, receptor accumulations reappear in the central regions of about one-third of the muscle fibers. This susceptibility of junctional AChR aggregates lasts only a short while during development of the neuromuscular junction. By the time of birth, end plate receptor aggregates have become resistant to these treatments.     

Desensitization of the muscarinic receptor controlling action potentials of sympathetic ganglion cells in bullfrogs

The desensitization of the muscarinic receptor, of which activation is known to depress the ionic K⁺ and Ca²⁺ currents generated during action potentials of bullfrog sympathetic ganglion cells, was studied. The depression of these voltage-dependent K⁺ and Ca²⁺ currents by muscarinic action of acetylcholine (ACh) was gradually restored to a certain extent when an application of ACh was sustained. After removal of ACh, the sensitivity of the muscarinic receptor was still depressed for an extended period, while resting and action potentials were apparently observed to be of normal level and size, respectively. These results indicate that desensitization of muscarinic receptors developed during a sustained application of ACh. It was suggested that the muscarinic receptor controlling these voltage-dependent currents of ganglion cells may be part of receptor-ionic channel complex (RICC) the nature of which was comparable to that of the RICC of the nicotinic receptor of the end-plate.     

Development of the neuromuscular junction in the chick embryo: The number,distribution, and stability of acetylcholine receptors

The number, distribution, and stability of skeletal muscle acetylcholine receptors during development of the neuromuscular junction in the chick embryo were studied. The distribution and turnover of receptors labeled with ¹²⁵I-labeled α-bungarotoxin were determined by quantitative autoradiography on individual teased muscle fibers. Each posterior latissimus dorsi muscle fiber, which in the adult is singly innervated, has a high density of acetylcholine receptors at a single spot from embryonic Day 10 through hatching. The spots stain more intensely than elsewhere for acetylcholinesterase and are assumed to be end plates. The receptors at these spots are presumed to be junctional receptors. The junctional receptor density remains constant at 10⁴/μm² from embryonic Day 14 through adult life, although the area of the junction increases 40-fold. In contrast, the extrajunctional receptor density drops precipitously from 250/μm² on Day 14 to only 10/μm² on Day 19. This decrease in extrajunctional receptor density can be prevented by chronic paralysis with curare. The rate of autoradiographic grain loss from junctional and extrajunctional regions after a pulse injection of ¹²⁵I-labeled α-bungarotoxin indicates that both classes of embryonic receptors turn over at the same rate ($\text{t}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 30}\text{hr}$).     

Role of the NO-cGMP cascade in regulation of L-type Ca2+ currents in isolated cardiomyocytes

Regulatory mechanisms of voltage-dependent L-type Ca²⁺ channels involving the cyclic nucleotide system of mammalian cardiomyocytes have been studied. Activation of cGMP-dependent phosphorylation in the presence of 1 mM arginine in all experimental media resulted in inhibition of amplitudes of basal L-type Ca²⁺ currents in rat ventricular myocytes. Effects of compounds regulating the activity of different compoments of the NO-cGMP cascade on L-type Ca²⁺ currents were investigated. It was found that endogenous (arginine, 5 mM) and exogenous (sodium nitroprusside, 1 mM) NO sources decreased the Ca²⁺ current amplitude by 30 ± 10%. The nonspecific NO synthase blocker 7NI (2 μM) abolished the effect of arginine, while the soluble guanylyl cyclase blocker ODQ (50 μM) eliminated the effects of both arginine and sodium nitroprusside. The fact that inhibitory effects of arginine, sodium nitroprusside and 8Br-cGMP disappeared in the presence of the protein kinase G blocker KT5823 (0.5, 1 μM) provides direct evidence in favor of activating effect of these compounds on PKG-dependent phosphorylation. Inhibition of L-type Ca²⁺ currents can also be due to activation of phosphodiesterase II. However, the selective phosphodiesterase II blocker EHNA (30 μM) failed to abolish inhibitory effects of arginine and sodium nitroprusside on Ca²⁺ currents. Isoproterenol (0.1 μM)-activated L-type Ca²⁺ currents were only partly blocked by acetylcholine (0.1 mM). Contrary to basal currents, the NO-cGMP cascade agonists arginine and sodium nitroprusside (SNP), like 8Br-cGMP, had no effect on isoproterenol-induced currents. Full inhibition of isoproterenol-induced currents was achieved through combination of acetylcholine with NO-cGMP cascade agonists.     

Acetylcholine receptor kinetics at slow fiber neuromuscular junctions

Acetylcholine receptors in slow fiber neuromuscular junctions of garter snake (sp. Thamnophis) produced synaptic responses that were more complicated than those observed from twitch fibers. Although the slow fiber miniature end plate currents decayed monoexponentially with time, both the current fluctuations spectrum and the voltage jump end plate current required two temporal components for good theoretical fits. This behavior was accurately accounted for by a generalized version of the three-state kinetic model by del Castillo and Katz. Application of the model allowed not only the rate of channel closing to be estimated, but also the rate of channel opening (from the closed state with acetylcholine bound) and the apparent rate of acetylcholine unbinding from the receptor. The results suggest that at the peak of the miniature end plate current local receptor saturation occurs.     

Actions of phencyclidine and its thienylpyrrolidine analogue on synaptic transmission and axonal conduction in the central nervous system of the cockroach Periplaneta americana

The effects of phencyclidine (PCP) and its thienylpyrrolidine analogue (TCPY) were tested on conduction processes in the isolated axon of giant interneurone 2 (GI 2) of the cockroach Periplaneta americana and on binding of [³H]PCP and [¹²⁵I]α-bungarotoxin to membranes from Periplaneta brain and nerve cord. Their actions on synaptic transmission between cercal sensory neurones and GI 2, where acetylcholine is the likely neurotransmitter, were also examined. PCP suppressed both sodium and potassium currents in the axonal membrane at 5.0 × 10^?4 M. Block was reversible on rebathing the axon in normal saline. TCPY exerted similar effects on the axon, though at slightly higher concentrations. Excitatory postsynaptic potentials (EPSPs) recorded from GI 2 in response to electrical stimulation of cercal nerve XI were progressively blocked by 5.0 × 10^?4 M PCP following a brief initial enhancement (?10%) of EPSP amplitude. The depolarizing response of GI 2 to ionophoretically applied acetylcholine was also blocked at this concentration, indicating a postsynaptic action of PCP at the acetylcholine receptor-ion channel of GI 2. TCPY also blocked synaptic transmission at synapses between cercal afferents and GI 2, but, in contrast to the actions of PCP, EPSP block was accompanied by depolarization. PCP and TCPY inhibited [³H]PCP binding to nerve cord and brain membranes with multiple affinities, suggesting multiple molecular targets. They also modified aspects of the kinetics of [¹²⁵I]α-bungarotoxin binding to the nicotinic acetylcholine receptor in these membranes and enhanced conversion of the receptor to the high affinity desensitized state. At higher concentrations they also inhibited [¹²⁵I]α-bungarotoxin binding. PCP was more potent than TCPY in inhibiting [³H]PCP binding but less potent on [¹²⁵I]α-bungarotoxin binding. Thus PCP and TCPY, which are structurally very similar, interact with several molecular targets in insect neuronal membranes, including sodium and potassium channels and acetylcholine receptors.     

The effects of amantadine on liposomally reconstituted nicotinic acetylcholine receptor

The effects of amantadine on liposomally reconstituted nicotinic acetylcholine receptor function were studied. At 1 × 10^?4M, the drug blocked 85% of the carbamylcholine-induced cation influx into liposomes, but left 90% of the αbungarotoxin binding intact. In addition, amantadine was shown to be a non-competitive inhibitor of membrane bound acetylcholinesterase. These experiments are relevant to the mechanism of action of amantadine at the motor end plate, where it produces electrophysiological changes compatible with an inhibition of cholinergic agonist mediated ion flux.     

Endogenous low molecular weight inhibitors of cholinergic binding sites

Endogenous low molecular weight compounds which inhibit ligand binding to nicotinic acetylcholine receptors of neuronal membranes have been isolated from insect nervous tissue. Two distinct heat-stable, cationic inhibitory compounds with molecular weights of about 700-500 Da and below 500 Da have been identified. The active material was found to competitively inhibit [¹²⁵I]α-bungarotoxin and [³H]acetylcholine binding in a reversible, dose dependent manner. Comparative binding studies revealed that the active material also inhibits [¹²⁵I]α-bungarotoxin and [³H]acetylcholine binding in vertebrate brain, but not in the electric tissue of Torpedo. These results suggest that the endogenous inhibitors may function as modulators specific for neuronal acetylcholine receptors.     

Patch-clamp recordings of spiking and nonspiking interneurons from rabbit olfactory bulb slices: GABA- and other transmitter receptors

Summary Transmitter receptor ion channels from previously identified rabbit olfactory bulb neurons were studied by using a thin slice preparation in combination with patch-clamp measurements. PG cells, which closely resembled previously described periglomerular interneurons in their morphology, responded to microapplication of GABA, acetylcholine, norepinephrine and glycine with the activation of distinct ionic currents. JG cells, which belong either to the class of short axon cells or external tufted cells, never showed GABA responses. In mitral cells ionic currents activated by GABA, acetylcholine, norepinephrine and glutamate could be elicited. Further measurements of GABA-activated currents of PG cells were made and indicated that these cells expressed two different types of GABA receptors: one which showed fast desensitization with a decay time constant of about 5 s, and one which slowly desensitized with a decay time constant of about 20–30 s. Both types were completely inhibited by bicuculline methiodide (50 M). GABA receptors were not blocked by Zn²⁺ (0.1 mM). From the dose-response relationship of the peak GABA-activated currents, an apparent dissociation constant of 50 M was derived. From single channel measurements in excised outside-out patches, a single channel conductance of GABA-activated Cl^– currents of 24 pS was obtained during continuous application of the agonist. Single channel events had a mean open time of 1.9 ms.     

Blocking action of Nephila clavata spider toxin on ionic currents activated by glutamate and its agonists in isolated hippocampal neurons

The blocking action ofNephila clavata spider neurotoxin, or JSTX, on ionic currents activated by L-glutamate and its agonists when applied to the membrane of neurons isolated from the rat hippocampus was investigated using a concentration clamp technique. Crude JSTX venom was found to block L-glutamate-, quisqualate, and kainate-activated ionic currents induced by activating non-N-methyl-D-aspartate (non-NMDA) membrane receptors. Following the effects of JSTX, ionic currents activated by L-glutamate and its agonists declined to 34–36% of their initial value with no recovery during JSTX washout. An active fraction of JSTX at concentrations of 10^–4–10^–5 produced almost total but partially reversible blockade of ionic currents. The action of JSTX became less effective during depolarization. The concentration dependence of JSTX-induced blockade of kainate-activated ionic currents was investigated and the velocity constants of interaction between the toxin and glutamate receptors obtained. It is postulated that JSTX interacts with chemically-operated non-NMDA ionic channels, blocking their transition into a number of their possible open states.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 21, No. 2, pp. 152–160, March–April, 1989.     

Binding-gating coupling in a nondesensitizing α7 nicotinic receptor: A single channel pharmacological study

The highly conserved αLys145 has been suggested to play an important role in the early steps of activation of the nicotinic acetylcholine receptor (nAChR) by acetylcholine. Both macroscopic and single channel currents were recorded in the slowly desensitizing mutants L248T- and K145A-L248T-α7 receptors expressed in Xenopus oocytes. On ACh-evoked currents, substitution of Lys145 by alanine showed the same effects that in wild type receptors: moderately decreased gating function and a more-than-expected loss of ACh potency, thus validating the experimental model. Single channel analysis quantitatively agreed with macroscopic data and revealed that impaired gating function in the double mutant α7K145A/L248T is the consequence of a slower opening rate, β. Several nicotinic agonists were also studied, showing important features. Particularly, dimethylphenylpiperazinium (DMPP), acting as an antagonist in α7K145A, became a full agonist in α7K145A/L248T. Single channel analysis of DMPP-evoked currents showed effects of Lys145 removal similar to those observed with ACh. Data suggest that α7Lys145 facilitates the early steps of channel activation. Moreover, the slowly desensitizing mutant α7L248T could be an interesting tool for the study of channel activation in α7 receptors. Nevertheless, its extensively altered pharmacology precludes the simple extrapolation of pharmacological data obtained in singly mutated α7 receptors.     

Effect of High-Frequency Stimulation on the Time Course of End-Plate Currents

We investigated the effects of repetitive high-frequency (10 sec^-1) nerve stimulation on the time course of evoked and miniature end-plate currents (EPC and mEPC, respectively) in the frog neuromuscular junction. The data obtained indicate that at a physiological Ca²⁺ level in the bath medium, 10 sec^-1 stimulation results in prolongation of the growth phase of multiquantum EPC without any effect on the mEPC time course. It is concluded that timing of acetylcholine quantum secretion may be affected by high-frequency stimulation.     

Specific [3H]quinuclidinyl benzilate binding activity in Digitonin-solubilized preparations from bovine brain

Receptors for the specific muscarinic radioligand [³H]quinuclidinyl benzilate ([³H]QNB) were solubilized by digitonin from a particulate preparation of bovine brain without significant alteration in binding affinities for muscarinic antagonists. Electron microscopy and sucrose density gradient sedimentation analysis confirmed the solubility of these receptors in aqueous solutions of digitonin. Equilibrium and kinetic studies of [³H]QNB binding to solubilized receptors indicated that binding was stereoselective and was blocked by muscarinic compounds. These tests permit tentative identification of digitonin-solubilized [³H]QNB binding sites as muscarinic acetylcholine receptors. Digitonin-solubilized receptors were homogeneous with respect to sedimentation behavior and binding affinities for agonist and antagonist drugs, unlike membrane-bound receptors. Enzyme digestion studies and treatment with group-specific reagents indicated that muscarinic receptors are proteins whose binding activity could be disrupted by reduction with dithiothreitol or by modification of sulfhydryl residues.     

Cardiomyocyte-like cells differentiated in vitro from embryonic carcinoma cells P19 are characterized by functional expression of adrenoceptors and Ca2+ channels

Summary P19 embryonal carcinoma cells were differentiated via embryolike aggregates (embryoid bodies) into spontaneously beating myocytes. During the whole process of differentiation the functional expression of cardiac-specific receptors and ionic channels was characterized by measuring the chronotropic reactivity, action potentials, and ionic currents in response to various cardioactive drugs. Positive chronotropic effects obtained at different maximal effective concentrations of adrenoceptor-mediated agonists indicated differential adrenoceptor expression during the in vitro development of cardiomyocyte-like cells. No cardiac-specific response was obtained with the muscarinic cholinoceptor agonist carbachol. Single beating cells were enzymatically isolated and investigated by the patch-clamp technique. Pacemaker action potentials similar to those of embryonal cardiomyocytes exhibited amplitudes ranging from 50 to 85 mV. The action potentials were synchronous to the mechanical contractions and, comparable to the chronotropic effects, were modulated by BayK 8644, isradipine, and adrenaline. The functional expression of L-type Ca²⁺ channels was demonstrated by the Ca²⁺ channel blockers isradipine, nisoldipine, gallopamil, and diltiazem causing negative chronotropic responses, as well as by the Ca²⁺ channel activator BayK 8644 causing positive chronotropic responses. These effects gradually increased with time of differentiation. The expression of L-type Ca²⁺ channels and of nicotinic acetylcholine receptors was confirmed in voltage-clamp experiments. The study demonstrates that P19 embryonal carcinoma cells can be induced to differentiate into cardiomyocyte-like cells comparable to embryonal and neonatal heart cells lacking the muscarinic cholinoceptor response only.     

The modulation of calcium currents by the activation of mGluRs

Glutamatergic transmission in the central nervous system (CNS) is mediated by ionotropic, ligand-gated receptors (iGluRs), and metabotropic receptors (mGluRs). mGluRs are coupled to GTP-binding regulatory proteins (G-proteins) and modulate different second messenger pathways. Multiple effects have been described following their activation; among others, regulation of fast synaptic transmission, changes in synaptic plasticity, and modification of the threshold for seizure generation. Some of the major roles played by the activation of mGluRs might depend on the modulation of high-voltage-activated (HVA) calcium (Ca²⁺) currents. Some HVA Ca²⁺ channels (N-, P-, and Q-type channels) are signaling components at most presynaptic active zones. Their mGluR-mediated inhibition reduces synaptic transmission. The interference, by agonists at mGluRs, on L-type channels might affect the repetitive neuronal firing behavior and the integration of complex events at the somatic level. In addition, the mGluR-mediated effects on voltagegated Ca²⁺ signals have been suggested to strongly influence neurotoxicity. Rather different coupling mechanisms underlie the relation between mGluRs and Ca²⁺ currents: Together with a fast, membrane-delimited mechanism of action, much slower responses, involving intracellular second messengers, have also been postulated. In the recent past, the relative paucity of selective agonists and antagonists for the different subclasses of mGluRs had hampered the clear definition of the roles of mGluRs in brain function. However, the recent availability of new pharmacological tools is promising to provide a better understanding of the neuronal functions related to different mGluR subtypes. The analysis of the mGluR-mediated modulation of Ca²⁺ conductances will probably offer new insights into the characterization of synaptic transmission and the development of neuroprotective agents.     

Pharmacology of the neuronal nicotinic acetylcholine receptor of cultured Kenyon cells of the honeybee, <Emphasis Type="Italic">Apis mellifera</Emphasis>

We investigated the pharmacology of the nicotinic acetylcholine receptor of honeybee Kenyon cells, a subset of olfactory interneurons, which are crucial for olfactory learning and memory. Whole-cell currents were recorded using patch-clamp techniques. Pressure application of agonists induced inward currents in cultured Kenyon cells at holding potentials of –110 mV. Acetylcholine or carbamylcholine were full agonists, nicotine, epibatidine and cytisine were only partial agonists. Coapplications of these partial agonists with acetylcholine reduced the current amplitude. The most efficient antagonists were dihydroxy--erythroidine (EC₅₀=0.5 pmol·l^–1) and methyllycaconitine (EC₅₀=24 pmol·l^–1). The open channel blocker mecamylamine, d-tubocurarine and hexamethonium were rather weak blockers of the honeybee nicotinic response. Bath applications of the muscarinic antagonist atropine inhibited nicotinic currents dependent on concentration (EC₅₀=24.3 mol·l^–1). Muscarine, pilocarpine or oxotremorine (1 mmol·l^–1) did not induce any measurable currents. The non-cholinergic drugs strychnine, bicuculline and picrotoxin partially and reversibly blocked the acetylcholine-induced currents. Our results indicate the expression of only one nicotinic acetylcholine receptor subtype in cultured Kenyon cells. Muscarinic as well as non-cholinergic antagonists also inhibit the receptor function, distinguishing the honeybee nicotinic receptor from the typical nicotinic receptor of vertebrates and from many described insects receptors.     

Nerve growth factor receptors on PC12 cells: Evidence for two receptor classes with differing cytoskeletal association

PC12, an NGF responsive cell line, exhibits two classes of NGF receptors which we designate “Fast” and “Slow.” Fast receptors, accounting for 75% of specific NGF binding, are distinguished by their rapid rates for association and dissociation of ¹²⁵I-NGF. At 37°C, binding of ¹²⁵I-NGF to Fast receptors is 5-fold more rapid than to Slow receptors and dissociation of ¹²⁵I-NGF from Fast receptors is 40-fold more rapid than from Slow receptors. No evidence was obtained for a ligand-induced conversion of receptors from Fast to Slow characteristics. Scatchard analysis of binding experiments indicates that PC12 cells possess 60,000 specific receptors for NGF of which 15,000 are of the Slow class. Despite having very different kinetic constants, Slow and Fast receptors have similar equilibrium binding constants (about 2 × 10^?10 M) due to cancelling effects of differing association and dissociation rates. Brief digestion of PC12 cells with trypsin before addition of NGF inactivates essentially all Fast receptors without significantly affecting Slow receptors. Therefore Fast and Slow classes of receptors must exist prior to addition of NGF, and the observed receptor heterogeneity is not due to ligand-induced changes. ¹²⁵I-NGF bound to Slow receptors is preferentially associated with preparations of Triton X-100 insoluble cytoskeletons, while ¹²⁵I-NGF bound to Fast receptors is solubilized by this procedure. Cytoskeletally associated NGF is almost exclusively associated with the extranuclear cytoskeletal matrix rather than with the nucleus itself. Preparation of nuclei by various methods suggests that the presence of contaminating cytoskeletal elements should be considered in evaluating the existence of translocation and binding of NGF to the nucleus. Inhibition of endocytotic internalization of NGF either by lowering of temperature to O°C or by preincubation of cells with sodium azide in medium lacking glucose does not reduce the slowly released component of bound NGF, nor alter its cytoskeletal association. The possible functional roles of Slow and cytoskeletal receptors are discussed.