Biomedical subjects

J C Hardwick

Publications and source records attributed to J C Hardwick.

12 recordsLinked to original sources

PubMed1997

Transmitter release differs at snake twitch and tonic endplates during potassium-induced nerve terminal depolarization.

Twitch and tonic muscle fibers of snake skeletal muscle differ in their synpatic as well as mechanical properties. These experiments were aimed at detemining the basis of the difference in vesicular release properties of nerve terminals at twitch and tonic endplates. Miniature endplate currents (MEPCs) were recorded from voltage-clamped garter snake muscle fibers depolarized by high K+ in either a control Ca2+ or high-Ca2+ solution. MEPC frequency increased at twitch and tonic endplates and remained elevated for 8 h during depolarization in control Ca2+. At twitch endplates depolarized in the presence of high Ca2+, an increase in MEPC frequency was followed by a progressive decline. In contrast, MEPC frequency remained elevated in high Ca2+ at tonic endplates. The observed decrease in MEPC frequency at depolarized twitch endplates in high Ca2+ was not a function of the level of depolarization or initial MEPC frequency, nor was it due to a reduction in MEPC amplitude and loss of MEPCs in baseline noise. An optical assay of presynaptic function in which the activity-dependent dye FM1-43 was used confirmed that quantal releases differs at twitch and tonic endplates. Most twitch nerve terminals were labeled by FM1-43 during prolonged depolarization with control Ca2+ or after brief depolarization with high Ca2+. In contrast, the number of twitch nerve terminals and the degree to which they were stained was greatly reduced after prolonged exposure to high K+ and high Ca2+, whereas depolarized tonic endplates were well stained by FM1-43 during brief and prolonged exposure to high Ca2+. FM1-43 staining also revealed variable levels of quantal release between individual boutons at twitch endplates after prolonged depolarization in high-Ca2+ solution. The observed reduction in presynaptic function at twitch nerve terminals after prolonged depolarization in high-Ca2+ solution was reversible and therefore not due to irreversible damage to terminal boutons. MEPC frequency increased at both twitch and tonic endplates when either Sr2+ or Ba2+ was substituted for high Ca2+ during K(+)-induced depolarization. Over time, in Sr2+ or Ba2+ solutions, MEPC frequency remained elevated at tonic endplates but declined at twitch endplates with a time course similar to that observed in high Ca2+. MEPC amplitudes at both endplates remained constant. We conclude that the regulation of quantal release differs in nerve terminals innervating twitch and tonic endplates and postulate that differential intraterminal accumulation of Ca2+ may underlie the observed difference in presynaptic function.

Animals↗

PubMed1996

Activation of the protein phosphatase calcineurin during carbachol exposure decreases the extent of recovery from end-plate desensitization.

1. Our previous studies demonstrated that protein kinase C (PKC) activity is required for acetylcholine (ACh) sensitivity to recover fully at snake twitch fiber end plates after prolonged exposure to carbachol. In the present studies, we have investigated whether protein phosphatase(s), activated during carbachol exposure, dephosphorylated critical membrane proteins, which required rephosphorylation by PKC to maintain end-plate sensitivity. End-plate sensitivity was assessed from measurements of miniature end-plate currents (MEPCs) and carbachol-activated currents (EPCCARBS). Conductance of ACh-activated channels was determined from patch-clamp recordings of single-channel currents. 2. Pretreatment of snake muscle preparations with the protein kinase inhibitor staurosporine (0.5 microM), followed by a 10-min exposure to 540 microM carbachol, reduced mean MEPC amplitudes to values 30-40% less than those recorded before carbachol exposure. Conversely, at control end plates exposed to carbachol, the mean MEPC amplitude was reduced by only approximately 5% compared with precarbachol values. This staurosporine-induced decrease in ACh sensitivity could be prevented by pretreatment with the protein phosphatase 2B (calcineurin) inhibitor deltamethrin (0.5 microM), whereas okadaic acid (5 microM) and calyculin A (0.5 microM), inhibitors of protein phosphatases 1 and 2A, had no effect. 3. After a 10-min exposure to 540 microM carbachol, EPCCARB amplitudes (produced by local superfusion with 20 microM carbachol) were significantly smaller at staurosporine-treated end plates than at control end plates. In contrast, the EPCCARB amplitude recorded from end plates pretreated with both deltamethrin and staurosporine was not significantly different from that recorded at control end plates. 4. Substitution of 10 mM Mn2+ for external Ca2+ during the exposure to 540 microM carbachol prevented the decrease in MEPC amplitude recovery at staurosporine-treated end plates. These results suggested that the alteration in sensitivity at staurosporine-treated end plates was calcium dependent. 5. At control end plates, a single population of ACh-activated channels (45-50 pS) is observed both before and after a 10-min exposure to 540 microM carbachol. Conversely, at staurosporine-treated end plates, after exposure to carbachol, a second population of small-conductance (25-30 pS) ACh-activated channels is present in addition to the predominant 45- to 50-pS ACh-activated channels. In preparations pretreated with both deltamethrin and staurosporine, after carbachol exposure, there was a significant decrease in the frequency of small-conductance ACh-activated channels. Deltamethrin treatment alone produced no small-conductance channels before or after a 10-min exposure to 540 microM carbachol. Also, no small-conductance ACh-gated channels were recorded at PKC-inhibited end plates after carbachol exposure either with pretreatment with 10 microM cyclosporin A (another inhibitor of calcineurin) or with the substitution of 10 mM Mn2+ for Ca2+ during the 10-min agonist exposure. 6. We propose that during prolonged exposure to the nicotinic agonist carbachol, calcium influx through ACh-gated channels elevates the level of ionized calcium at the inner surface of the post-junctional membrane and that this local rise in intracellular calcium activates the calcium-dependent phosphatase calcineurin. Dephosphorylation of some key membrane protein by calcineurin leads to a decrease in the extent of recovery from desensitization. Under normal conditions, this process is effectively reversed by PKC activity and end-plate sensitivity recovers fully. However, when PKC is inhibited, the extent of recovery of end-plate sensitivity is decreased, and associated with this decrease is the presence of small-conductance ACh-activated channels not normally recorded at snake twitch fiber end plates.

Animals↗

PubMed1995

Evidence for afferent fiber innervation of parasympathetic neurons of the guinea-pig cardiac ganglion.

The present study was done to establish whether peptidergic afferent inputs can modulate parasympathetic neurons of the guinea-pig cardiac ganglion. Whole mount preparations from the guinea-pig heart were utilized to localize afferent terminals by immunohistochemistry and for intracellular recordings from individual neurons in situ. Action potentials could be elicited by both intracellular current injection and stimulation of interganglionic fiber bundles. Two types of neuron, phasic (95%) and tonic (5%) as defined by their firing properties, were observed. High frequency (5-10 Hz) interganglionic fiber stimulation produced a calcium-dependent, slow depolarization in many cells which was not blocked by 100 microM hexamethonium or 1 microM atropine. A prolonged depolarization was also produced by local application of capsaicin (1 mM), which releases substance P and CGRP from afferent nerve terminals. Microinjection of the mammalian tachykinins substance P, neurokinin A and neurokinin B (all at 100 microM), also produced a slow depolarization. Application of specific agonists for the tachykinin receptor subtypes indicated that these neurons express both NK2 and NK3 receptors. Individual cells were filled with neurobiotin to examine their morphology and the preparations were counter-stained for SP-like immunoreactivity. The results demonstrated that SP-positive fibers are found in close apposition to both phasic and tonic neurons. From these results, we suggest that the parasympathetic neurons of the guinea-pig cardiac ganglion receive inputs from peptidergic, afferent fibers and that this input provides a pathway for potential local reflex control of cardiac function.

Animals↗

PubMed1995

Necessity of protein kinase C activity for maintenance of acetylcholine receptor function at snake twitch fibre endplates.

1. The extent of recovery of endplate sensitivity following a 5 or 10 min exposure to carbachol was determined from measurements of miniature endplate current (m.e.p.c.) amplitudes in voltage-clamped snake twitch fibre endplates. M.e.p.c. amplitude recovery was dependent on the carbachol concentration (0.27-5.4 mM) and duration of application. Staurosporine pretreatment (0.5 microM for approximately 15 min) further decreased the extent of m.e.p.c. amplitude recovery. 2. The decrease in m.e.p.c. amplitude at control endplates exposed to high concentrations of agonist (5.4 mM carbachol for 10 min) was due to an apparent decrease in postsynaptic receptor density, not to a change in the conductance of the acetylcholine (ACh)-activated channels. 3. Pretreatment with either 1 microM lavendustin A or 50 microM KN-62 had no effect on m.e.p.c. amplitude recovery, whereas pretreatment with either 0.5 microM staurosporine, 50 microM sphingosine, or 0.5 microM calphostin C significantly reduced m.e.p.c. amplitude recovery following carbachol exposure. 4. Sphingosine and staurosporine produced a concentration-dependent decrease in the extent of m.e.p.c. amplitude recovery, but had no effect on m.e.p.c. characteristics in the absence of carbachol. In addition, this decrease in m.e.p.c. amplitude was not due to the presence of a subpopulation of small amplitude m.e.p.cs. 5. Prolonged treatment (18-20 h) of muscles with 200 nM phorbol 12-myristate 13-acetate (PMA), to down regulate protein kinase C, resulted in a significant reduction in m.e.p.c. amplitudes following exposure to carbachol. Conversely, treatment with 200 nM 4 alpha PMA, an inactive analogue, had no effect on m.e.p.c. amplitude recovery. 6. Only large amplitude ACh-activated channels (~50 pS) were recorded from fibres either in the presence of 50 micro M sphingosine or from fibres chronically exposed to PMA. However, following recovery from a 10 min exposure to 540 micro M carbachol, both small conductance (-25 pS) and large conductance ACh-activated channels were recorded in both sphingosine- and phorbol-treated preparations. The conductance of these two populations of channels was virtually identical to those seen in staurosporine treated fibres following carbachol exposure.7. We conclude that protein kinase C is required for full recovery of AChR sensitivity following carbachol-induced receptor inactivation. Exposure to high concentrations of agonist for prolonged periods appears to result in the inactivation of a subpopulation of receptors. These receptors must be replaced or reactivated by a process involving protein kinase C. When this phosphorylation step is inhibited, the AChRs remain in an activatable form, but with a reduced conductance.

Animals↗

PubMed1995

Requirement of a colchicine-sensitive component of the cytoskeleton for acetylcholine receptor recovery.

1. The effect of colchicine treatment on acetylcholine receptor function was examined in potassium depolarized, voltage-clamped snake twitch fibre endplates. Receptor function was assessed by analysis of miniature endplate currents (m.e.p.c.) as well as acetylcholine (ACh)-induced single channel currents. 2. Pretreatment of snake muscle fibres with colchicine (10 microM to 100 microM) for 16-18 h had no effect on m.e.p.c. amplitude or decay rates. At higher concentrations (1 mM), there was a slight decrease in the average m.e.p.c. amplitude. 3. Colchicine produced a concentration-dependent decrease in the extent of m.e.p.c. amplitude recovery following a 10 min exposure to 540 microM carbachol. Exposure of 100 microM colchicine-treated preparations to 0.5 microM staurosporine further reduced the extent of m.e.p.c. amplitude recovery following carbachol exposure. 4. The decrease in m.e.p.c. amplitude following carbachol exposure was not due to a shift in the m.e.p.c. reversal potential. In addition, the distribution of m.e.p.c. amplitudes remained unimodal in both control and colchicine (100 microM)-treated preparations following carbachol exposure. 5. In addition to the normal, large conductance (approximately 48 pS) ACh-activated channels, a population of small conductance (approximately 29 pS) channels was observed in colchicine-treated preparations following exposure to carbachol. In preparations treated with both colchicine and staurosporine and then exposed to carbachol, the conductance of these small channels was identical to that of colchicine or staurosporine alone. 6. We suggest that prolonged exposure of snake twitch fibre endplates to agonist results in the activation and desensitization of ACh receptors. Furthermore, we propose that for a subpopulation of the inactivated receptors, restoration of function requires both the integrity of a subsynaptic cytoskeletal component and phosphorylation by a staurosporine-sensitive protein kinase. One plausible mechanism is that some receptors become destabilized in the membrane and phosphorylation of a cytoskeletal component, whose distribution may depend on an intact microtubular system, is required to re-anchor these receptors. If this anchoring process is inhibited either by disruption of the cytoskeleton with colchicine, or inhibition of the kinase by staurosporine, these receptors remain activatable, but have a reduced conductance.

Alkaloids↗

PubMed1993

Mechanism of staurosporine-induced decrease in acetylcholine receptor recovery from desensitization.

1. Previously, we showed in voltage-clamped snake twitch muscle fibres that the extent of recovery of the nicotinic acetylcholine (ACh) receptor from carbachol-induced desensitization is reduced by pretreatment with the protein kinase inhibitor staurosporine. The present studies were undertaken to determine the mechanism underlying the staurosporine-induced inhibition of recovery. 2. Pretreatment with 0.5 microM staurosporine significantly decreased the extent of recovery of spontaneous miniature endplate current (m.e.p.c.) amplitudes in preparations exposed to 540 microM carbachol. The decrease in recovery of m.e.p.c. amplitude by staurosporine was dependent on the duration of carbachol exposure. No significant decrease in m.e.p.c. amplitude was observed with a 1 min exposure to agonist, whereas a significant decrease in recovery was seen with agonist exposures between 5-10 min. Further, the effect of staurosporine pretreatment on ACh receptor recovery was long-lasting such that m.e.p.c. amplitude remained decreased for at least 60 min. 3. Estimation of mean channel conductance by noise analysis during local perfusion of 20 microM carbachol demonstrated a decrease in conductance from 52 pS to 23 pS in staurosporine-treated preparations following recovery from desensitization. Staurosporine treatment in the absence of desensitization did not alter the mean channel conductance. 4. A single population of ACh-activated single channel currents with a conductance of 45-49 pS was recorded in cell-attached patches from enzymatically cleaned endplates in control and staurosporine-treated preparations not exposed to carbachol. 5. At staurosporine-treated endplates exposed to carbachol and then allowed to recover, a population of small conductance (23 pS) channels was observed. These channels were not normally seen in control preparations which had undergone carbachol-induced desensitization and recovery.6. We suggest that the decrease in m.e.p.c. amplitude observed following recovery from desensitization in staurosporine-treated endplates results from the activation of a mixture of small and large conductance ACh receptor channels.

Alkaloids↗

PubMed1993

Necessity of divalent cations for recovery from carbachol-induced nicotinic acetylcholine receptor inactivation at snake twitch fibre endplates.

1. Previous studies demonstrated that elevation of the extracellular calcium concentration during a prolonged exposure to a high concentration of carbachol reverses the staurosporine-induced decrease in the extent of endplate resensitization in voltage-clamped snake twitch fibres. The present studies were designed to establish the site, specificity and potential mechanisms by which calcium could reverse the effects of staurosporine on acetylcholine receptor recovery. 2. Pretreatment of potassium-depolarized muscle preparations with 0.5 microM staurosporine, followed by a 10 min incubation with 540 microM carbachol, produced a significant decrease in the recovery of miniature endplate current (m.e.p.c.) amplitudes. Raising the extracellular calcium concentration from 1 mM to 10 mM during the agonist application reversed this inhibition. In addition, a brief (3 min) incubation in an elevated calcium (10 mM) solution in staurosporine-treated preparations previously exposed to 540 microM carbachol also reversed the reduction in m.e.p.c. amplitude. 3. Substitution of calcium with 10 mM strontium had no effect on the staurosporine-induced decrease in m.e.p.c. amplitude, whereas 10 mM magnesium partially substituted for calcium. Inclusion of (+)-tubocurarine (13 microM) in the 10 mM calcium solution to prevent the influx of calcium through agonist-activated channels did not prevent the reversal of the staurosporine-induced decrease in m.e.p.c. amplitudes. This suggested that the site of action of calcium on endplate resensitization was extracellular. 4. Analysis of acetylcholine (ACh)-induced single channel currents demonstrated that a population of small conductance channels seen only in the staurosporine-treated preparations following carbachol exposure, was still present in staurosporine-treated preparations incubated in 540 microM carbachol solution containing 10 mM calcium. Thus the effect of calcium on ACh receptor recovery was not due to the conversion of the small conductance channels to the normal, large conductance channels.5. Removal of calcium from the extracellular solution with the magnesium concentration unchanged,resulted in no change in the extent of m.e.p.c. amplitude recovery, nor did it alter the ability of staurosporine to inhibit recovery. Removal of both calcium and magnesium resulted in a significant decrease in the extent of recovery and staurosporine produced no additional decrease. This decrease in m.e.p.c. recovery in the divalent cation-free solution was not associated with a change in mean channel conductance as determined by noise analysis.6. Based on the results from these experiments, we suggest that, with prolonged exposure to agonist,some ACh receptors at the endplate become irreversibly inactivated. For full recovery of endplate sensitivity to occur, inactivated ACh receptors must be replaced. Part of the replacement ACh receptors appear to be recruited from a readily available but not previously activated pool and this process is dependent on extracellular calcium and/or magnesium. Thus, elevation of extracellular calcium may be able to reverse the apparent decrease in endplate sensitivity in staurosporine-treated fibres following carbachol exposure by increasing the number of functional receptors at the endplate.

Alkaloids↗

PubMed1993

Quantal transmitter release at snake twitch and tonic muscle fibres during prolonged nerve terminal depolarization.

1. Miniature endplate currents (MEPCs) were recorded in vitro from voltage-clamped twitch and tonic muscle fibres in costocutaneous muscles of the garter snake, Thamnophis. Recordings were made from fibres in a control sodium-containing solution and then during exposure to an isotonic potassium solution containing either 1.0 mM calcium and 4.2 mM magnesium or 3.6 mM calcium. The experiments were done at two levels of external calcium in order to demonstrate that the change in MEPC frequency was calcium dependent. During the initial exposure to the isotonic potassium solutions, the MEPC frequency was increased manyfold at both twitch and tonic fibres, but it declined progressively with continued exposure. MEPCs were recorded from both fibre types throughout a 20 h exposure to the isotonic potassium solution with 1 mM calcium, but no MEPCs were recorded at most twitch endplates after approximately 6 h in the isotonic potassium solution containing 3.6 mM calcium. In contrast, MEPCs were still present at tonic fibre endplates after 20 h in the isotonic potassium solution containing 3.6 mM calcium. 2. After 30 min in the isotonic potassium solution with 1 mM calcium, the MEPC amplitude recorded from both fibre types was approximately twice that in the control sodium-containing solution. At tonic endplates, the MEPC amplitude was also twofold greater in the isotonic potassium solution with 3.6 mM calcium than in sodium-containing solution. In contrast, after 30 min in the isotonic potassium solution containing 3.6 mM calcium, the MEPC amplitude at twitch endplates was similar to that in control solution. 3. In both fibre types, MEPC amplitude decreased progressively with continued exposure to the isotonic potassium solutions. The progressive decrease in MEPC amplitude was not due to a gradual decrease in postsynaptic sensitivity to acetylcholine. 4. The effects of high potassium were reversible as MEPCs were recorded at twitch fibre endplates in preparations which were returned to the control sodium-containing solution after a 20 h exposure to the isotonic potassium solution containing 3.6 mM calcium. 5. Ultrastructural examination showed that after a 6 h exposure to the isotonic potassium solutions most nerve terminals innervating twitch fibre endplates were devoid of synaptic vesicles whereas at the same time many synaptic vesicles were present in nerve terminals innervating tonic fibre endplates. Surprisingly, numerous synaptic vesicles were present in nerve terminals innervating either fibre type in muscle preparations exposed to the isotonic potassium solutions for 20 h.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗

PubMed1992

Galanin stimulates phosphatidylinositol turnover in cardiac tissue of the mudpuppy.

A galanin-like peptide has been found in the parasympathetic fibers innervating the mudpuppy myocardium and direct application of galanin produces hyperpolarization of atrial myocytes and a decrease in twitch tension. In the present study, atrial and ventricular strips were incubated with galanin and then evaluated for changes in either phosphatidylinositol turnover or cyclic nucleotide levels. Galanin caused a significant and concentration-dependent increase in phosphatidylinositol turnover in both atrial and ventricular tissue. However, galanin had no effect on cyclic AMP or cyclic GMP levels in either basal or adrenergically-stimulated preparations. These results suggest that the galanin-induced cardio-inhibition in the mudpuppy may be mediated, at least in part, by a change in phosphatidylinositol turnover.

Animals↗

PubMed1992

Aminergic and peptidergic elements and actions in a cardiac parasympathetic ganglion.

Correlated histochemical, immunocytochemical, and electrophysiological experiments have been undertaken to identify putative neurotransmitter-neuromodulator substances in cells and fibers in the parasympathetic cardiac ganglion of the mudpuppy, Necturus maculosus, and to determine the action of these agents on the properties of the parasympathetic postganglionic neurons. The mudpuppy cardiac ganglion contains two neuron types: large parasympathetic postganglionic neurons and smaller intrinsic neurons initially identified as small intensely fluorescent cells. We have shown that the postganglionic neurons contain both acetylcholine and a galanin-like neuropeptide. Also, we have demonstrated that the intrinsic neurons contain a number of different biogenic amines such as dopamine and serotonin, as well as neuropeptides including a substance P-like peptide and a galanin-like peptide. The results of these studies indicate that the anatomical and histochemical organization of the mudpuppy cardiac ganglion is more complex than that seen in other amphibians and is very similar to that found in most mammalian species. Previously, we showed that galanin has actions that make it of interest as a potential inhibitory neurotransmitter in the mudpuppy cardiac ganglion. Galanin hyperpolarizes and decreases membrane excitability in most parasympathetic neurons. Here we show that galanin initiates membrane hyperpolarization by activating a voltage- and time-dependent potassium conductance. We also present the initial results of ongoing studies which indicate that calcitonin gene-related peptide can depolarize some of the parasympathetic neurons as well as evidence that serotonin initiates depolarization in many parasympathetic neurons. This serotonin-induced depolarization consists of an initial transient depolarization followed by a longer, more slowly developing depolarization. Action potential activity is stimulated during the initial period of depolarization, but depressed during the later, slow depolarization. The results of these electrophysiological experiments suggest that many of the bioactive substances that have been identified in the different cells and nerve fibers within the cardiac ganglion affect the excitability of the postganglionic neurons. In conclusion, we suggest that the results of the studies summarized in this review demonstrate that the cardiac ganglion in the mudpuppy is not simply a relay station. Rather, the cardiac ganglion has a complex organization and exhibits a diversity of physiological responses, indicating that it very likely is another site of integration for control of cardiac function.

Animals↗

PubMed1991

Staurosporine inhibits the extent of acetylcholine receptor recovery from carbachol-induced desensitization in snake twitch fibres.

1. The effect of the protein kinase inhibitor, staurosporine, on the extent and time course of recovery following carbachol-induced desensitization was studied in snake twitch-muscle fibres maintained in an isotonic potassium propionate solution and voltage-clamped to +30 mV. 2. Pretreatment with staurosporine (0.5 microM) decreased the extent of recovery of spontaneous miniature endplate current (m.e.p.c.) amplitudes following desensitization by a sustained application of 540 microM carbachol. Recovery was inhibited by approximately 50% without altering the time course of m.e.p.c. recovery. 3. Staurosporine also produced a concentration-dependent (10 nM to 0.5 microM) decrease in the amplitude of a second carbachol-induced current, following a wash period, as compared to the amplitude of the current produced by the initial carbachol application. Pretreatment with 0.5 microM K252a, another wide spectrum protein kinase inhibitor, also decreased the extent of recovery of the response to a second carbachol application following desensitization. 4. Staurosporine pretreatment (0.5 microM) had no effect on either the kinetics of receptor-channel gating or the initial endplate sensitivity to agonist. This was determined by comparing the amplitude of the carbachol (540 microM)-induced currents and the amplitude and decay rate of m.e.p.cs in control and staurosporine-treated fibres. 5. Staurosporine had no effect on the time course of desensitization onset produced during the initial application of 540 microM carbachol or the depth of desensitization produced by the end of a 2-3 min exposure to 540 microM carbachol.6. Elevation of the external calcium concentration from 1 to 10mM during the 540 microM carbachol application completely antagonized the decreased extent of recovery of m.e.p.c. amplitude produced by pretreatment with 0.5 microM staurosporine.7. We suggest that phosphorylation of a population of acetylcholine receptors is required for complete recovery from desensitization, and that staurosporine inhibits the protein kinases responsible for this phosphorylation.8. We further propose that a transient increase in intracellular calcium, produced by an increase in calcium influx through agonist-activated endplate channels, stimulates additional protein kinase activity, which in turn, antagonizes the effect of staurosporine-treatment on recovery.

Alkaloids↗

PubMed1989

Extracellular ATP stimulates norepinephrine uptake in PC12 cells.

This study examined the effects of extracellular ATP on norepinephrine (NE) uptake, using PC12 cells as a model of noradrenergic neurons. Previous experiments with synaptosomes led to the hypothesis that extracellular ATP can regulate NE uptake via an ecto-protein kinase. In the present study, we examined the high-affinity uptake of NE (referred to as uptake 1) in PC12 cells in the presence of varying concentrations of extracellular ATP. In the presence of Ca2+, low concentrations of ATP (0.1 microM) increased uptake 1 by approximately 36%. This increase could be mimicked by adenosine-5'-O-(3-thiotriphosphate) tetralithium salt (ATP gamma S), an analogue of ATP which can be utilized by protein kinases, and not by 5'-adenylylimidodiphosphate tetralithium salt, a nonhydrolyzable analogue of ATP, GTP, ADP, and adenosine also had no effect on uptake 1. Preincubation of the cells with NE and ATP gamma S, followed by washing and assaying NE uptake 30 min later, resulted in a persistent increase in uptake 1. Similar pretreatment with ATP did not show this increase; however, simultaneous pretreatment with ATP and ATP gamma S blocked the activation produced by ATP gamma S alone. Kinetic analysis showed that ATP gamma S pretreatment produces an increase in the Vmax of uptake 1 without altering the apparent Km for NE. These results support the hypothesis that extracellular ATP can regulate NE uptake via an ecto-protein kinase.

Adenosine Triphosphate↗