ATP-induced Depolarization Research Articles

Mechanisms of activation of nucleus accumbens neurons by cocaine via sigma-1 receptor–inositol 1,4,5-trisphosphate–transient receptor potential canonical channel pathways

Cocaine promotes addictive behavior primarily by blocking the dopamine transporter, thus increasing dopamine transmission in the nucleus accumbens (nAcc); however, additional mechanisms are continually emerging. Sigma-1 receptors (σ1Rs) are known targets for cocaine, yet the mechanisms underlying σ1R-mediated effects of cocaine are incompletely understood. The present study examined direct effects of cocaine on dissociated nAcc neurons expressing phosphatidylinositol-linked D1 receptors. Endoplasmic reticulum-located σ1Rs and inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) were targeted using intracellular microinjection. IP3 microinjection robustly elevated intracellular Ca2+ concentration, [Ca2+]i. While cocaine alone was devoid of an effect, the IP3-induced response was σ1R-dependently enhanced by cocaine co-injection. Likewise, cocaine augmented the [Ca2+]i increase elicited by extracellularly applying an IP3-generating molecule (ATP), via σ1Rs. The cocaine-induced enhancement of the IP3/ATP-mediated Ca2+ elevation occurred at pharmacologically relevant concentrations and was mediated by transient receptor potential canonical channels (TRPC). IP3 microinjection elicited a slight, transient depolarization, further converted to a greatly enhanced, prolonged response, by cocaine co-injection. The cocaine-triggered augmentation was σ1R-dependent, TRPC-mediated and contingent on [Ca2+]i elevation. ATP-induced depolarization was similarly enhanced by cocaine. Thus, we identify a novel mechanism by which cocaine promotes activation of D1-expressing nAcc neurons: enhancement of IP3R-mediated responses via σ1R activation at the endoplasmic reticulum, resulting in augmented Ca2+ release and amplified depolarization due to subsequent stimulation of TRPC. In vivo, intra-accumbal blockade of σ1R or TRPC significantly diminished cocaine-induced hyperlocomotion and locomotor sensitization, endorsing a physio-pathological significance of the pathway identified in vitro.

Purinergic modulation of area postrema neuronal excitability in rat brain slices

ATP has been shown to excite neurons in various regions of the central nervous system. Whereas immunohistochemical studies show P2X receptors in the area postrema, the responsiveness of area postrema neurons to extracellular ATP has not been studied. To investigate the effects of purinoceptor activation on area postrema neuronal excitability, we performed whole-cell recordings from area postrema neurons in rat brain slices. Most area postrema neurons responded to ATP application, and most responses were excitatory. Voltage-clamp recordings showed three different types of response: (1) a postsynaptic or extrasynaptic excitatory response (inward currents; n = 26/51 cells), (2) a presynaptic excitatory response (increased frequency of miniature excitatory postsynaptic currents with only a small direct postsynaptic current; n = 24/51 cells, or (3) a postsynaptic inhibitory response (outward current; n = 1/51). The excitatory responses were found in both of the two major electrophysiological cell classes, i.e. cells displaying I h and cells not displaying I h, while the inhibitory responses were found in only cells not displaying I h. Current-clamp recordings showed ATP-induced depolarization ( n = 13/15) or hyperpolarization ( n = 2/15) of membrane potential that modulated the frequency of action potentials. In the presence of CNQX, mEPSCs were abolished and bath-applied ATP did not generate mEPSCs, indicating that glutamate release was facilitated by the activation of presynaptically located ATP receptors. Our pharmacological results from studies with ATP, αβme-ATP, βme-ATP and PPADS indicate that the post- and/or extrasynaptic responses are most likely mediated by P2X 7 receptors and/or receptors composed of P2X 2 and P2X 5 subunits. We conclude that half of the presynaptic responses are most likely mediated by P2X 7 receptors and/or receptors composed of P2X 2 and P2X 5 subunits while the others also contain P2X 1 subunits. It is well known that P2X 7 subunit forms only homomultimeric P2X receptors. Finally, the present study suggests that purinoceptor activation may contribute to the control of several autonomic functions by area postrema neurons.

Effects of adenosine and ATP on the membrane potential and synaptic transmission in neurons of the rat locus coeruleus.

Effects of adenosine (Ado) and adenosine 5'-triphosphate (ATP) on the membrane potential and synaptic transmission in neurons of the rat locus coeruleus (LC) were examined, in vitro. Ado (30-300 microM) produced a hyperpolarizing response and inhibited spontaneous firing activity in neurons of the rat LC. Ado decreased input resistance of LC neurons. The Ado-induced hyperpolarization reversed polarity near the equilibrium potential of K+ (EK). Ado (100-300 microM) depressed both excitatory postsynaptic potential (EPSP) and inhibitory postsynaptic potential (IPSP). Ado (300 microM) did not alter the hyperpolarization induced by norepinephrine (30 microM). N6-Cyclopentyladenosine (CPA, 100 microM), an A1 receptor agonist, also produced a hyperpolarizing response and depressed both the EPSP and IPSP. Another A1 receptor agonist, adenosine amine congener (ADAC, 30 microM) also produced a hyperpolarizing response and consistently depressed the EPSP and IPSP. Application of ATP (100 microM) to LC neurons caused a depolarizing response associated with an increase in the firing rate of spontaneous action potential in LC neurons. The ATP-induced depolarization was accompanied by an increased input resistance and reversed polarity at--91 mV. ATP (100 microM) consistently depressed the IPSP, while it did not change the amplitude of the EPSP in a majority of neurons. alpha, beta-Methylene ATP (alpha, beta-meATP, 30 micro/M), a P2 receptor agonist, mimicked these effects of ATP. Adenosine 5'-(beta, gamma-imido) triphosphate (AMP-PNP, 100 microM), a non-metabolizable analogue of ATP, produced a depolarizing response in LC neurons, but it produced no obvious depression of the EPSP and IPSP. These results suggest that Ado and ATP cause inhibitory and excitatory modulation, respectively, of neuronal activity and synaptic transmission in the rat LC.

Activation of ATP receptor increases the cytosolic Ca 2+ concentration in ventral tegmental area neurons of rat brain

ATP increased the cytosolic Ca 2+ concentration ([Ca] i) in neurons of ventral tegmental area acutely dissociated from rat brain. The ATP response was dependent on external Ca 2+ and Na +, and was blocked by voltage-dependent Ca 2+ channel blockers. The results suggest that the ATP-induced depolarization increases Ca 2+ influx via voltage-gated Ca 2+ channels resulting in the increase in [Ca] i.

Activation of ATP receptor increases the cytosolic Ca 2+ concentration in nucleus accumbens neurons of rat brain

ATP increased the cytosolic Ca 2+ concentration ([Ca] i) in nucleus accumbens neurons acutely dissociated from rat brain. The ATP response was dependent on external Ca 2+ and Na +, and was blocked by voltage-dependent Ca 2+ channel blockers. The results suggest that the ATP-induced depolarization increases Ca 2+ influx resulting in the increase in [Ca] i.

Effects of ATP and derivatives on neuropile glial cells of the leech central nervous system.

We investigated the effects of ATP (adenosine 5'-triphosphate) and derivatives on leech neuropile glial cells, focusing on exposed glial cells. ATP dose-dependently depolarized or hyperpolarized neuropile glial cells in situ as well as exposed neuropile glial cells. These potential shifts varied among cells and repetitive ATP application did not change their amplitude, duration or direction. In exposed neuropile glial cells, ATP most frequently induced a Na(+)-dependent depolarization and decreased the input resistance. The agonist potency ATP > ADP (adenosine 5'-diphosphate) > AMP (adenosine 5'-monophosphate) > adenosine indicates that P2 purinoceptors mediate this depolarization. The P2Y agonist 2-methylthio-ATP mimicked the ATP-induced depolarization, whereas the P2Y antagonist PPADS (pyridoxal-phosphate-6-azophenyl-2', 4'-disulphonic acid) reduced it. P2X agonists were without effect. Because the P1 antagonist 8-SPT (8-(p-sulphophenyl)-theophylline) also depressed ATP-induced depolarizations and some ATP-insensitive glial cells responded to adenosine, we suggest coexpression of metabotropic P2Y and P1 purinoceptors. The ATP-induced depolarization requires activation of Na(+) channels or nonselective cation channels, whereas the ATP-induced hyperpolarization indicates activation of K(+) channels. ATP also increased the intracellular Ca(2+) concentration ([Ca(2+)](i)), that is independent of Ca(2+) influx but reflects intracellular Ca(2+) release possibly triggered by IP(3) formation. ADP and AMP also increased [Ca(2+)](i), but were less efficient than ATP; adenosine and 2-methylthio-ATP did not affect [Ca(2+)](i). In view of the mobilization of intracellular Ca(2+), ATP is clearly different from other leech neurotransmitters, because it enables intracellular Ca(2+) signaling without causing prominent changes in glial membrane potential. Thus disturbance of the extracellular microenvironment and the demand for metabolic energy are minimized.

Extracellular ATP raises cytosolic calcium and activates basolateral chloride conductance in Necturus proximal tubule.

1. Extracellular nucleotides modulate ionic transport mechanisms in various epithelia. In the present study, we investigated the effects of extracellular ATP on the intracellular free Ca+2 concentration ([Ca2+]i) and electrophysiological properties of Necturus maculosus proximal convoluted tubule (PCT). 2. ATP raised [Ca2+]i in microdissected fura-2-loaded PCTs (half-maximal effect, approximately mumol 1(-1) ATP). The initial ATP-induced changes in [Ca2+]i were not blunted by the removal of external Ca2+ nor by the presence of Ca2+ channel blockers, but were abolished by thapsigargin and suramin. The sequence for the potency of various agonists on [Ca2+]i was 2-methylthioATP (2MeSATP) = ADP = ATP >> UTP, 2',3',-O-(4-benzoilbenzoil) ATP (BzATP), alpha, beta-methylene ATP (AMPCPP), adenosine. 3. In vivo electrophysiological measurements showed that 100 mumol 1(-1) peritubular ATP added to a Ringer solution reduced the basolateral cell membrane potential (Vm) and increased the cell membrane input conductance. In a low Cl- solution, this ATP-induced depolarization was enhanced. These effects were inhibited by 1 mmol l-1 SITS, consistent with the activation of a basolateral Cl- conductance. 4. The ATP-induced change in Vm was reproduced by ADP but not by UTP or adenosine, and was prevented by suramin. 5. The ATP-induced membrane depolarization was not influenced by thapsigargin, BAPTA AM, or staurosporine and was not reproduced by manoeuvres increasing [Ca2+]i or intracellular cAMP content. 6. We conclude that, in Necturus PCT, a P2y receptor mobilizes Ca2+ mainly from intracellular pools and increases a basolateral Cl- conductance, GCl. The activation of GCl occurs by a mechanism which is not related either to an increase in [Ca2+]i or cAMP content, or to PKC activation.

Agonist-induced activation of a non-selective ion current in glomerular endothelial cells

The control of intracellular calcium activity ([Ca2+]i) and membrane voltage (Vm) play an important role in regulating functions of glomerular endothelial cells (GEC). We investigated the effect of extracellular ATP on the intracellular [Ca2+]i, Vm and ion conductances in GEC. ATP (100 mumol/liter) induced a rapid increase of [Ca2+]i in GEC from 20 +/- 6 to 442 +/- 84 nmol/liter, which was followed by a sustained Ca2+ plateau of 112 +/- 29 nmol/liter. In a bath solution with a low extracellular Ca2+ concentration the ATP-induced [Ca2+]i peak was still present, but the [Ca2+]i plateau was completely prevented. In 186 experiments with the patch clamp technique the addition of ATP (1 to 100 mumol/liter) to GEC induced a transient small hyperpolarization, which was followed by a depolarization. During the ATP-induced depolarization an increase of the whole cell conductance was found. The Ca2+ ionophore A23187 (10 mumol/liter) mimicked the effect of ATP on Vm. Reduction of the extracellular Ca2+ to 1 mumol/liter itself depolarized GEC reversibly from -88 +/- 2 to -60 +/- 12 mV and increased the ATP-induced depolarization to -18 +/- 3 mV. In the absence of Na+ in the bathing solution (replacement by NMDG+) ATP induced only an attenuated depolarization and no inward current was activated. Flufenamate (100 mumol/liter), a blocker of non-selective ion channels inhibited the ATP-induced depolarization of Vm significantly by 58 +/- 13%, whereas nicardipine (10 mumol/liter) or amiloride (10 mumol/liter) had no effect. Our data indicate that the resting Vm of GEC cells is almost completely dominated by K+ conductances and that ATP activates a Ca2+ dependent non-selective ion conductance in GEC.

UTP and ATP induce different membrane voltage responses in rat mesangial cells.

UTP and ATP induce different membrane voltage responses in rat mesangial cells. Recent studies have indicated that UTP and ATP might modulate mesangial cell function in a different manner. Here we compared the effect of UTP and ATP on membrane voltage (Vm) and ion currents in mesangial cells in primary culture, and we examined whether different nucleotide receptors are involved. In patch-clamp experiments in the fast whole cell configuration, UTP (in contrast to ATP) caused a sustained and concentration-dependent depolarization (half-maximal effective dose, 10(-5) M), but ATP caused only a transient depolarization. During the depolarization, UTP induced a sustained increase of the whole cell conductance (Gm), whereas ATP induced only a transient increase of Gm. When cells were dialyzed with Cs2SO4 and extracellular Cl- was replaced by 145 mM sodium gluconate, addition of UTP or ATP (both 10(-4) M) did not significantly increase Gm. Addition of ATP in the presence of UTP caused an additional depolarization by 5 mV, which was followed by a hyperpolarization by 21 mV. Repetitive application of ATP led to an attenuation of the ATP-induced depolarization. Then, in the presence of ATP, UTP still induced a significant depolarization by 10 mV. Suramine and reactive blue 2 did not inhibit the depolarization induced by UTP, but these inhibited the Vm response to ATP. In microfluorescence experiments, UTP and ATP caused a concentration-dependent increase of the intracellular calcium activity ([Ca2+]i) in mesangial cells. Application of both UTP and ATP had no additive effect on [Ca2+]i. The results suggest that mesangial cells possess, in addition to P2y purinoceptors, separate nucleotide receptors for UTP.

Extracellular ATP regulates glomerular endothelial cell function.

1. Glomerular endothelial cells form the inner part of the filtration barrier and are involved in pathophysiological processes in the glomerulum. New techniques for culturing glomerular endothelial cells have been established recently. The effect of extracellular ATP on membrane voltage and intracellular calcium activity was examined in bovine glomerular endothelial cells (GEC) in culture. 2. Membrane voltage was measured with the patch clamp technique in the fast whole cell configuration. GEC possess a stable membrane voltage of -88 mV. ATP induced a small transient hyperpolarization, which was followed by a depolarization. The ATP-induced depolarization was significantly inhibited by flufenamate, a blocker of non-selective ion channels. 3. The intracellular calcium activity [Ca2+]i was measured in single cells with the fura-2 technique. ATP stimulated an increase of [Ca2+]i. The increase of [Ca2+]i was biphasic with an initial peak followed by a sustained plateau. The [Ca2+]i peak was still present in an extracellular Ca(2+)-free solution, whereas the plateau was inhibited. 4. The order of potency of different purine nucleotides in stimulating [Ca2+]i and inositol formation was UTP = ATP > ATP-gamma-S > 2-methylthio ATP > [alpha,beta-CH2]ATP. 5. The data indicate that ATP regulates membrane voltage and [Ca2+]i in glomerular endothelial cells by a P2y2 receptor.

The use of antagonists to characterize the receptors mediating depolarization of the rat isolated vagus nerve by alpha, beta-methylene adenosine 5'-triphosphate.

1. We have previously found that the P2x-purinoceptor agonist, alpha, beta-methylene adenosine 5'-triphosphate (alpha, beta-methylene ATP), depolarizes the rat cervical vagus nerve, measured with a 'grease-gap' extracellular recording technique. This effect was attenuated by the P2 purinoceptor antagonist, suramin. In the present study we have investigated in more detail the antagonism produced by suramin and have also investigated the actions of two other putative P2 purinoceptor antagonists, cibacron blue and pyridoxal-phosphate-6-azophenyl-2', 5'-disulphonic acid (iso-PPADS). Furthermore, we have studied the interactions between suramin and cibacron blue or iso-PPADS in an attempt to determine whether these antagonists act at a common receptor site. 2. Suramin (1 x 10(-5)-1 x 10(-4) M) produced reversible, concentration-related rightward displacements of the concentration-effect curve to alpha, beta-methylene ATP. Schild analysis of this antagonism yielded a pA2 value of 5.90 with a slope value of 0.47. 3. Cibacron blue (3 x 10(-5)-1 x 10(-4) M) also antagonized depolarizations induced by alpha, beta-methylene ATP. The antagonistic effects of cibacron blue were slow to reach equilibrium but could be readily reversed on washout. At low concentrations for antagonism, cibacron blue (1 x 10(-5) M and 3 x 10(-5) M) produced enhancement of the maximal response to alpha, beta-methylene ATP. At the highest concentration tested (1 x 10(-4) M) the concentration-effect curve to alpha, beta-methylene ATP was shifted to the right in a parallel manner, yielding a pKB estimate of 4.96. 4. Iso-PPADS (1 X 10-6 1 X 10-5- M) produced a concentration-related depression in the maxima ofthe concentration-effect curves to alpha,beta-methylene ATP. Analysis of these data by a double reciprocal plot yielded a pKB estimate of 6.02. This profile of insurmountable antagonism could not be attributed to irreversible binding of iso-PPADS to the receptor since the effect of iso-PPADS could be reversed on washing, albeit slowly.5. In the presence of suramin (1 x 10-4 M), cibacron blue (1 x 10-4 M) produced no further rightward displacement of the alpha,beta-methylene ATP concentration-effect curve. The mean agonist concentration ratios in the presence of suramin or cibacron blue alone (11.7 and 10.3, respectively) were not significantly different from the mean concentration-ratio in the presence of both antagonists (11.8). This finding suggests that high concentrations of alpha,beta-methylene ATP activate a receptor population which is resistant to blockade by either antagonist.6. The antagonistic effect of iso-PPADS (1 x 10-5 M) was partially attenuated by suramin (1I x 10-4 M).It is possible that this interaction reflects a slow dissociation of iso-PPADS from the receptor with which suramin and alpha,beta-methylene ATP interact.7. Suramin, cibacron blue or iso-PPADS had no marked effect on depolarization produced by 5-hydroxytryptamine (5-HT, 1 x 10-7-3 x 10-5 M), indicating their specificity in antagonizing responses to alpha, beta-methylene ATP.8. Responses to alpha,beta-methylene ATP were not antagonized by 8-para-sulphophenyltheophylline (3 x 10-5M), ondansetron (1 x 10-7 M), bicuculline (1 x I0-5 M), phentolamine (1 X 10-6 M) or hexamethonium(1 X 10-4 M), which are antagonists at P1-purinoceptors, 5-HT3 receptors, GABAA receptors, a-adrenoceptors and nicotinic cholinoceptors, respectively, thereby excluding the involvement of these receptors.Indomethacin (3 X 10-6 M) had no effect on responses to alpha,beta-methylene ATP.9. The results obtained with three purinoceptor antagonists confirm and extend our original supposition that alpha,beta-methylene ATP-induced depolarization of the rat vagus nerve is mediated predominantly via P2 purinoceptors, thought to be of the P2,X subtype. The finding that responses induced by high concentrations of agonist were resistant to blockade by suramin and cibacron blue, but could be attenuated by iso-PPADS, adds further weight to our speculation that the purinoceptor population in the rat vagus nerve is heterogeneous.

Effect of extracellular ATP on contraction, cytosolic calcium activity, membrane voltage and ion currents of rat mesangial cells in primary culture

1. The effects of extracellular ATP on contraction, membrane voltage (Vm), ion currents and intracellular calcium activity [Ca2+]i were studied in rat mesangial cells (MC) in primary culture. 2. Addition of extracellular ATP (10(-5) and 10(-4) M) to MC led to a cell contraction which was independent of extracellular calcium. 3. Membrane voltage (Vm) and ion currents were measured with the nystatin patch clamp technique. ATP induced a concentration-dependent transient depolarization of Vm (ED50: 2 x 10(-6) M). During the transient depolarization ion currents were monitored simultaneously and showed an increase of the inward- and outward current. 4. In a buffer with a reduced extracellular chloride concentration (from 145 to 30 mM) ATP induced a depolarization augmented to -4 +/- 4 mV. 5. ATP-gamma-S and 2-methylthio-ATP depolarized Vm to the same extent as ATP, whereas alpha,beta-methylene-ATP (all 10(-5) M) had no effect on Vm. 6. The Ca2+ ionophore, A23187, depolarized Vm transiently from -51 +/- 2 to -28 +/- 4 mV and caused an increase of the inward current. 7. The intracellular calcium activity [Ca2+]i was measured with the fura-2 technique. ATP stimulated a concentration-dependent increase of [Ca2+]i (ED50: 5 x 10(-6) M). The increase of [Ca2+]i was biphasic with an initial peak followed by a sustained plateau. 8. The [Ca2+]i peak was still present in an extracellular Ca(2+)-free buffer, whereas the plateau was abolished. Verapamil (10(-4) M) did not inhibit the [Ca2+]i increase induced by ATP. 9. The data indicate that extracellular ATP contracts MC and is able to increase [Ca2+]i by the release of Ca2+ from intracellular stores and recruitment from the extracellular space. In addition ATP depolarizes Vm of MC by activating a Cl- conductance. The ATP-induced depolarization is mediated by a P2y receptor.

Depolarization of rat locus coeruleus neurons by adenosine 5′-triphosphate

Intracellular recordings were performed in a pontine slice preparation of the rat brain containing the locus coeruleus. The enzymatically stable P 2-purinoceptor agonist α,β-methylene ATP increased the firing rate without altering the amplitude or shape of action potentials; the afterhyperpolarization following a spike was not changed either. When locus coeruleus neurons were hyperpolarized by current injection in order to prevent spontaneous firing, α,β-methylene ATP produced depolarization and a slight increase in the apparent input resistance. A combined application of kynurenic acid and bicuculline methiodide failed to alter the α,β-methylene ATP-induced depolarization, and tetrodotoxin only slightly depressed it. A gradual shift of the membrane potential by hyperpolarizing current injection led to a corresponding decrease, but no abolition or reversal of the α,β-methylene ATP effect. In the hyperpolarized region, the current-voltage curve of α,β-methylene ATP came into close approximation with, but did not cross, the control curve. Elevation of the external K + concentration, or the intracellular application of Cs + by diffusion from the microelectrode, depressed the response to α,β-methylene ATP; external tetraethylammonium was also inhibitory. External Ba 2+ and Cs + had no effect or only slightly decreased the α,β-methylene ATP-induced depolarization. A low Na +, or a low Ca 2+ high Mg 2+ medium, as well as the presence of Co 2+ in the medium, markedly reduced or even abolished the depolarization by α,β-methylene ATP. ATP itself did not produce consistent changes in the membrane potential or input resistance. However, in the presence of the P 1-purinoceptor antagonist 8-cyclopentyl-1,3-dipropylxanthine, ATP consistently increased the firing rate and evoked an inward current. In conclusion, P 2-purinoceptor activation appears to depolarize locus coeruleus neurons by inhibiting a persistent potassium current, and at the same time opening calcium-sensitive sodium channels or calcium-sensitive non-selective cationic channels.

Mechanism of extracellular ATP-induced depolarization in rat isolated ventricular cardiomyocytes

Adenosine triphosphate (ATP) is released during neural stimulation and cardiac hypoxia and several mechanisms of its action have been reported in different tissues. ATP stimulates P1 and P2 purinergic receptors; it also activates receptor-operated channels and increases membrane permeability to small ions. In single rat ventricular cells under whole-cell patch-clamp, a stepwise application of ATP in the micromolar range affects the resting potential and membrane currents through an entirely novel mechanism of action which involves several steps. Extracellular ATP induces an inward current and depolarization of the cell, leading to automaticity. The inward current is non-specific for cations, its reversal potential is around -5 mV. The conductance change evoked by ATP is suppressed by 4,4-diisothiocyanostilbene 2,2-disulphonic acid (DIDS) and low-chloride media and is prolonged by adding intracellular bicarbonate. These effects are specific for ATP in the presence of magnesium and are not evoked by a non-hydrolysable analogue of ATP or in the presence of vanadate. Other nucleotides are ineffective. We propose that ATP hydrolysis activates the chloride/bicarbonate (Cl-/HCO3-) exchanger. The induced local acidification could then increase intracellular free calcium and as a consequence, increases the sarcolemmal conductance. Thus, a sudden release of ATP in pathological conditions would induce a depolarization which could generate ventricular arrhythmias.

The properties of the ATP‐induced depolarization and current in single cells isolated from the guinea‐pig urinary bladder

1. The actions of exogenously applied ATP were investigated with the whole-cell patch clamp method in single cells isolated from guinea-pig urinary bladder with a modified concentration jump technique. 2. Rapid application of ATP (threshold ca. 100 nM) depolarized the cell membrane with superimposition of action potentials which was followed by transient hyperpolarization. In the presence of D600, the amplitude of the ATP-induced depolarization was a function of the ATP concentration (EC50: 0.5-1 microM). 3. ATP activated a dose-dependent inward current with a short latency (18 ms with 10 microM ATP; measured as the time between the start of application and 10% of the peak). The relationship of the peak current versus ATP concentration was well fitted by a Michaelis-Menten equation with a Hill coefficient (n) of 1.7 and a dissociation constant (Kd) of 2.3 microM. The current desensitized rapidly and the time course of desensitization was a function of the ATP concentration and could be fitted by two exponentials. 4. The reversal potential of the ATP-activated current was near 0 mV. Replacement of extracellular Na by other monovalent or divalent cations indicated that the current flows through nonselective cation channels. 5. alpha,beta-Methylene ATP also produced a dose-dependent inward current but was less potent than ATP (n: 1.6, Kd: 10.4 microM). alpha,beta-Methylene ATP blocked the response to ATP by desensitization of the receptor. 6. alpha,beta-Methylene ATP was 50-100 times more potent than ATP at eliciting a contractile response of strips of detrusor smooth muscle. 7. The relevance of the above results to the possible role of ATP as the fast excitatory transmitter is discussed.

Adenosine 5'‐triphosphate modulates membrane potassium conductance in guinea‐pig myenteric neurones.

1. Intracellular recordings were made from myenteric neurones isolated from the guinea-pig small intestine to study actions of adenosine 5'-triphosphate (ATP). ATP was applied by superfusion (10 nM-100 microM) or pressure ejection from ATP-containing glass pipettes. 2. Myenteric neurones have been classified into two groups: type I/S neurones and type II/AH neurones. ATP produced a membrane hyperpolarization in 80% of AH neurones and a membrane depolarization in 90% of S neurones in a dose-dependent manner. Adenosine caused responses similar to those induced by ATP in both AH and S neurones, but was less effective than ATP. 3. The ATP-induced hyperpolarization was associated with a fall in input resistance, but the ATP-induced depolarization was accompanied by an increase in input resistance. Both responses reversed in polarity near the potassium equilibrium potential (-84 to -87 mV) and the reversal potential varied with extracellular potassium concentration, as predicted by the Nernst equation. These results indicate that the hyperpolarization is due to an increase, while the depolarization is due to a decrease in potassium conductance. 4. Both the hyperpolarization and the depolarization induced by ATP persisted in calcium-free solution containing 1.2 mM-magnesium, but were markedly reduced or abolished in calcium-free solutions containing 3.7-10 mM-magnesium and by 1 mM-nickel or cobalt. Both responses to ATP persisted in tetraethylammonium (1-10 mM) or tetrodotoxin (1-3 microM)-containing solutions. 5. Quinine and quinidine (1-100 microM) reversibly depressed both the ATP-induced responses. Caffeine (100 microM), theophylline (100 microM) and 3-isobutyl-1-methylxanthine (1-10 microM) did not significantly affect the ATP-induced depolarization but did reversibly depress the ATP-induced hyperpolarization. 6. These results suggest that the ATP-induced hyperpolarization may be due to activation, and the ATP-induced depolarization to inactivation, of a calcium-sensitive potassium conductance.

Membrane potential responses to ATP applied by pressure ejection in the longitudinal muscle of chicken rectum.

1. Changes in membrane potential in response to local application of ATP by pressure ejection from a micropipette were recorded intracellularly from smooth muscle cells of the longitudinal muscle strip of chicken rectum. 2. The local application of ATP produced a membrane depolarization. The depolarizing response increased to a plateau of 33 mV with prolongation of the duration of pressure pulses which determines the amount of ATP ejected. The latency and the time required to reach a peak depolarization were not related to the pulse duration, and the shortest latency was 50 ms. 3. When the application of ATP was repeated at a short interval, the second and subsequent depolarizing responses were suppressed, and their latency and time to reach a peak were also increased; the muscle cells became desensitized to ATP. Recovery from the desensitization occurred slowly over a period of 60 s. 4. Electrotonic potentials decreased in amplitude and time course during an ATP-induced depolarization, indicating a decrease in membrane resistance. 5. The ATP-induced depolarization was longer in the latency than an excitatory junction potential (e.j.p.) elicited by electrical field stimulation of the intramural nerves. The other variables such as amplitude, time to reach the peak and duration could not be matched with those of the e.j.p. at the same time. 6. The e.j.p. decreased in amplitude and duration during the ATP-induced depolarization, and its initial amplitude and duration were restored immediately after termination of the ATP-induced depolarization, as in the case of electrotonic potentials. The ej.ps were recorded in the same manner as the control from the muscle cell which was in a state of desensitization to ATP. 7. The present results provide an argument against the hypothesis that ATP mediates the ej.p. in the rectum of the chicken.

Extracellular ATP induces a large nonselective conductance in macrophage plasma membranes.

Extracellular ATP in its tetra-anionic form (ATP4-) induces ion fluxes and membrane depolarization in the mouse macrophage-like cell line J774.2 and in resident mouse macrophages. We analyzed the effects of extracellular ATP4- by both patch-clamp and intracellular microelectrode techniques. Whole-cell patch-configuration membrane potential measurements on J774.2 cells revealed that ATP4- -induced depolarization occurred within 40 ms of pulsed application of ATP and was completely reversible. The depolarizations were accompanied by a dramatic increase in membrane conductance and showed no sign of adaptation to ATP over a period of 30 min. At 5 mM total ATP (ATPt) the whole-cell conductance was approximately 10 nS, and an upper limit of 20 pS for a single-channel conductance has been established. The reversal potential associated with the ATP-induced depolarization at asymmetric K+, Na+, Ca2+, and Cl- concentrations across the membrane was 0 mV. In patch-clamped cells depolarization was complete at 20 microM ATP4-, and repolarization from full depolarization occurred in approximately 5 s. In contrast, in intact cells measured by microelectrode impalement, complete depolarization occurred at approximately 2 mM ATP4- and repolarization was much slower (approximately 100 min). These findings indicate that the changes in intracellular ionic composition that occur after ATP treatment affect the rate of cell repolarization. At lower concentrations of ATP, potassium conductances modulated the depolarizing effect of ATP. ATP also depolarized mouse peritoneal macrophages, but a variant cell line (ATPR B2), derived from J774.2 cells by prolonged exposure to ATP, was insensitive to ATP. Our results provide a membrane electrophysiological description and analysis of a large nonselective plasma membrane conductance of macrophages induced by extracellular ATP.

Effects of α,β-methylene ATP on electrical responses produced by ATP and nerve stimulation in smooth muscle cells of the guinea-pig mesenteric artery

1. In smooth muscle cells of the guinea-pig mesenteric artery, alpha, beta-methylene ATP(mATP) depolarized the membrane and decreased the membrane resistance. 2. Desensitization with mATP inhibited the excitatory junction potential (e.j.p.) and ATP-induced depolarization, while desensitization with ATP inhibited the e.j.p. but not the mATP-induced depolarization. 3. After desensitization with mATP, the response to mATP recovered rapidly in comparison with the e.j.p. and the ATP-induced depolarization. 4. The results suggest that mATP inhibits the ATP-induced depolarization by acting sites other than the ATP-receptor.

Effects of α,β-methylene ATP on electrical responses produced by ATP and nerve stimulation in smooth muscle cells of the guinea-pig mesenteric artery

Abstract o 1. In smooth muscle cells of the guinea-pig mesenteric artery, α,β -methylene ATP(mATP) depolarized the membrane and decreased the membrane resistance. 2. Desensitization with mATP inhibited the excitatory junction potential (e.j.p.) and ATP-induced depolarization,while desensitization with ATP inhibited the e.j.p. but not the mATP-induced depolarization. 3. After desensitization with mATP, the response to mATP recovered rapidly in comparison with thee.j.p. and the ATP-induced depolarization. 4. The results suggest that mATP inhibits the ATP-induced depolarization by acting sites other thanthe ATP-receptor.