Wolfgang Nörenberg

Neuronal ATP receptors and their mechanism of action

ATP stores and supplies energy in neurons, but it also acts as a transmitter molecule. ATP activates a class of membrane receptors termed P2 purinoceptors. Based on the potencies of structural analogues of ATP, P2 purinoceptors in non-neuronal tissues were classified by classic pharmacological methods into two subtypes, P2x and P2y. Peter Illes and Wolfgang Nörenberg report that electrophysiological investigations indicate the presence of P2y-like purinoceptors on neurons. They describe two alternative ionic transduction mechanisms that may be activated by this receptor family.

Characterization and possible function of adenosine 5'-triphosphate receptors in activated rat microglia.

1 Purinoceptor agonist‐induced currents in untreated (proliferating) and lipopolysaccharide (LPS; 100 ng ml−1)‐treated (non‐proliferating) rat microglial cells in culture were recorded by the whole‐cell patch‐clamp technique. These cells have two preferred resting membrane potentials, one at − 35 mV and another one at − 70 mV. 2 Most experiments were carried out in non‐proliferating cells. ATP, ATP‐γ‐S and α,β‐MeATP (1–1000 μm in all cases) evoked an inward current at a holding potential of − 70 mV, followed, in some experiments, by an outward current. At − 70 mV 2‐methylthio ATP (1–1000 μm) evoked an inward current, whereas at − 35 mV it produced an outward current only. 3 When K+ was replaced in the pipette solution by an equimolar concentration of Cs+ (150 mm), the main outward component of the ATP‐γ‐S (10 μm) induced response disappeared. Instead, an inward current was obtained. Replacement of K+ by Cs+ did not affect the inward current evoked by 2‐methylthio ATP (300 μm). 4‐Aminopyridine (1–10 mm), however, almost abolished this current and unmasked a smaller outward current. 4 The rank order of agonist potency was 2‐methylthio ATP >ATP>α,β‐MeATP. Adenosine and UTP were inactive. Suramin (300 μm) and reactive blue 2 (50 μm) antagonized the effect of 2‐methylthio ATP (300 μm). 5 I–V relations were determined by delivering fast voltage ramps before and during the application of 2‐methylthio ATP (300 μm). In the presence of extra‐ (1 mm) and intracellular (150 mm) Cs+, the 2‐methylthio ATP‐evoked current crossed the zero current level near 0 mV. When both Cs+ (1 mm) and 4‐aminopyridine (1 mm) were present in the bath medium, the intersection of the 2‐methylthio ATP current with the zero current level was near − 75 mV. 6 2‐Methylthio ATP (1–1000 μm) induced the same inward current both in proliferating and non‐proliferating microglia. However, the depolarizing response to 2‐methylthio ATP (300 μm) was larger and longer‐lasting in the proliferating cells. When the free Ca2+ concentration in the pipettes was increased from the standard 0.01 to 1 μm, the amplitude and duration of this depolarization was increased in non‐proliferating cells. 4‐Aminopyridine (1 mm) enhanced the duration, but not the amplitude of responses. 7 ATP and its structural analogues stimulate microglial purinoceptors of the P2Y‐type. This leads to the opening of non‐selective cationic channels and potassium channels. Depending on the resting membrane potential, depolarization or hyperpolarization prevails. Although the inward current produced by 2‐methylthio ATP is of similar amplitude in proliferating and non‐proliferating microglia, the resulting depolarization is smaller in the latter cell type because of the presence of voltage‐sensitive, outwardly rectifying potassium channels.

Voltage‐dependent potassium channels in activated rat microglia.

1. Voltage‐dependent currents of untreated (proliferating) and lipopolysaccharide (LPS)‐treated rat microglial cells in culture were recorded using the whole‐cell patch‐clamp technique. 2. Membrane potentials showed prominent peaks at ‐35 mV and ‐70 mV. Membrane potentials of LPS‐treated cells alternated between the two values. This may be due to a negative slope region of the I‐V relation resulting in two zero current potentials. 3. From a holding potential of ‐70 mV, hyperpolarizing steps evoked an inwardly rectifying current both in proliferating and in LPS‐treated cells, while depolarizing steps below ‐50 mV evoked an outwardly rectifying current only in LPS‐treated microglia. The currents were K+ selective, as indicated by their reversal potential of approximately 0 mV in symmetric K+ concentrations (150 mM both intra‐ and extracellularly) and the reversal potential of the outward tail currents of approximately ‐90 mV at a normal extracellular K+ concentration (4.5 mM). 4. The activation of the outward current could be fitted by Hodgkin‐Huxley‐type n4 kinetics. The time constant of activation depended on voltage. 5. The inactivation of the inward and outward currents could be fitted by a single exponential. The time constant of the inward current inactivation was dependent on voltage, whereas the time constant of the outward current inactivation was virtually independent of voltage, except near the threshold of activation. Recovery of the outward from inactivation was slow and could be fitted by two exponentials. Responses to depolarizing steps were stable at 0.125 Hz, but greatly decreased from the first to the second pulse at 1 Hz. 6. The inactivation of the inward, but not of the outward, current disappeared in a low Na(+)‐containing medium (5 mM). The inward current was selectively inhibited by extracellular Cs+ and Ba2+. The outward current was selectively inhibited by Cd2+, 4‐aminopyridine and charybdotoxin. Replacement of intracellular K+ by an equimolar concentration of Cs+, and the extracellular application of tetraethylammonium and quinine inhibited both currents. 7. An increase of extracellular Ca2+ from 2 to 20 mM resulted in outwardly rectifying K+ channels activating at more positive potentials. Omission of Ca2+ from the extracellular medium had the opposite effect. When the intracellular free Ca2+ was increased from 0.01 to 1 microM, the outward current amplitudes were depressed. The Ca2+ ionophore A23187 had a similar effect. 8. LPS‐treated microglial cells possess inwardly and outwardly rectifying K+ channels. The physiological and pharmacological characteristics of these two channel populations are markedly different.(ABSTRACT TRUNCATED AT 400 WORDS)

Inflammatory stimuli induce a new K+ outward current in cultured rat microglia

Membrane currents of cultured rat microglia were recorded with the whole-cell patch clamp technique. Undifferentiated microglia express only inwardly rectifying K+ channels. However, treatment of the cells with bacterial lipopolysaccharide, interferon-gamma, or their incubation in hydrophobic teflon bags, procedures that promote microglial differentiation, induced the expression of an additional outward current. Cycloheximide prevented the development of this conductance indicating the synthesis of a new channel protein. The reversal potential of the outward current was near to the K+ equilibrium potential; the current was abolished by intracellular Cs+ or extracellular 4-aminopyridine, and was depressed by extracellular tetraethylammonium. Hence, the channels involved appear to be highly selective for K+; their possible function is a rapid termination of depolarizing shifts of the membrane potential.

Effect of adenosine and some of its structural analogues on the conductance of NMDA receptor channels in a subset of rat neostriatal neurones

1 In order to investigate the modulatory effects of adenosine on excitatory amino acid projections onto striatal medium spiny neurones, whole‐cell patch clamp experiments were carried out in rat brain slices. The effects of various agonists for P1 (adenosine) and P2 (ATP) purinoceptors and their antagonists were investigated. The A2A receptor agonist 2‐p‐(2‐carboxyethyl)phenethylamino‐5′‐N‐ethylcarboxamidoadenosine (CGS 21680; 0.1 μM), the A1 receptor agonist 2‐chloro‐N6‐cyclopentyladenosine (CCPA; 10 μM) and the non‐selective P1 purinoceptor antagonist 8‐(p‐sulphophenyl)‐theophylline (8‐SPT; 100 μM) did not alter the resting membrane potential, the threshold current necessary to elicit an action potential, the amplitude of spikes, their rise time, the amplitude of the afterhyperpolarization (AHP) and the time to peak of the AHP. 2 N‐methyl‐D‐aspartate (NMDA; 1–1000 μM) caused a concentration‐dependent inward current which was larger in the absence than in the presence of Mg2+ (1.3 mM). In a subset of striatal neurones, the current response to NMDA (10 μM) and the accompanying increase in conductance were both inhibited by CGS 21680 (0.01–1 μM). The effect of CGS 21680 (0.1 μM) persisted in the presence of tetrodotoxin (0.5 μM) or in a Ca2+‐free medium, under conditions when synaptically mediated influences may be negligible. 3 The A3 receptor agonist N6‐2‐(4‐aminophenyl)ethyladenosine (APNEA; 0.1–10 μM) also diminished the effect of NMDA (10 μM), while the A1 receptor agonists CCPA (0.01–10 μM) and (2S)‐N6‐[2‐endo‐norbornyl]adenosine [S(−)‐ENBA; 10 μM] as well as the endogenous, non‐selective P1 purinoceptor agonist adenosine (100 μM) were inactive. The endogenous non‐selective P2 purinoceptor agonist ATP (1000 μM) also failed to alter the current response to NMDA (10 μM). Adenosine (100 μM), but not ATP (1000 μM) became inhibitory after blockade of nucleoside uptake by S(4‐nitrobenzyl)‐6‐thioguanosine (NBTG; 30 μM). 4 8‐(p‐Sulphophenyl)‐theophylline (8‐SPT; 100 μM), as well as the A2A receptor antagonist 8‐(3‐chlorostyryl)caffeine (CSC; 1 μM) and the A1 receptor antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX) at 0.03, but not 0.003 μM abolished the inhibitory action of CGS 21680 (0.1 μM). None of these compounds altered the effect of NMDA (10 μM) by itself. DPCPX (0.03 μM) prevented the inhibition by APNEA (10 μM). 5 There was no effect of CGS 21680 (0.1 μM), when guanosine 5′‐O‐(3‐thiodiphosphate (GDP‐β‐S; 300 μM) was included in the pipette solution in order to block G protein‐mediated reactions. 6 In conclusion, adenosine receptors, probably of the A2A‐subtype, inhibit the conductance of NMDA receptor channels in a subset of medium spiny neurones of the rat striatum by a transduction mechanism which involves a G protein.

Coexistence of purino‐ and pyrimidinoceptors on activated rat microglial cells

Nucleotide‐induced currents in untreated (proliferating) and lipopolysaccharide (LPS; 100ngml−1) treated (non‐proliferating) rat microglial cells were recorded by the whole‐cell patch‐clamp technique. Most experiments were carried out on non‐proliferating microglial cells. ATP (100nm–1mm), ADP (10nm–10mm) and UTP (1μm–100mm), but not uridine (100μm–10mm) produced a slow outward current at a holding potential of 0mV. The effect of UTP (1mm) did not depend on the presence of extracellular Mg2+ (1mm). The outward current response to UTP (1mm) was similar in non‐proliferating and proliferating microglia. In non‐proliferating microglial cells, the ATP (10μm)‐induced outward current was antagonized by suramin (300μm) or reactive blue 2 (50μm), whereas 8‐(p‐sulphophenyl)‐theophylline (8‐SPT; 100μm) was inactive. By contrast, the current induced by UTP (1mm) was increased by suramin (300μm) and was not altered by reactive blue 2 (50μm) or 8‐SPT (100μm). The current response to UTP (1mm) disappeared when K+ was replaced in the pipette solution by an equimolar concentration of Cs+ (150mm). However, the effect of UTP (1mm) did not change when most Cl− was replaced with an equimolar concentration of gluconate− (145mm). The application of 4‐aminopyridine (1mm) or Cs+ (1mm) to the bath solution failed to alter the UTP (1mm)‐induced current. UTP (1mm) had almost no effect in a nominally Ca2+‐free bath medium, or in the presence of charybdotoxin (0.1μm); the inclusion of U‐73122 (5μm) or heparin (5mgml−1) into the pipette solution also blocked the responses to UTP (1mm). By contrast, the effect of ATP (10μm) persisted under these conditions. I‐V relations were determined by delivering fast voltage ramps before and during the application of UTP (1mm). In the presence of extracellular Cs+ (1mm) and 4‐aminopyridine (1mm) the UTP‐evoked current crossed the zero current level near−75mV. Omission of Ca2+ from the Cs+ (1mm)‐ and 4‐aminopyridine (1mm)‐containing bath medium or replacement of K+ by Cs+ (150mm) in the pipette solution abolished the UTP current. Replacement of GTP (200μm) by GDP‐β‐S (200μm) in the pipette solution abolished the current evoked by UTP (1mm). When the pipette solution contained Cs+ (150mm) instead of K+ and in addition inositol 1,4,5,‐trisphosphate (InsP3; 10μm), an inward current absolutely dependent on extracellular Ca2+ was activated after the establishment of whole‐cell recording conditions. This current had a typical delay, a rather slow time course and did not reverse its amplitude up to 100mV, as measured by fast voltage ramps. A rise of the internal free Ca2+ concentration from 0.01 to 0.5μm on excised inside‐out membrane patches produced single channel activity with a reversal potential of 0mV in a symmetrical K+ solution. The reversal potential was shifted to negative values, when the extracellular K+ concentration was decreased from 144 to 32mm. By contrast, a decrease of the extracellular Cl− concentration from 164 to 38mm did not change the reversal potential. Purine and pyrimidine nucleotides act at separate receptors in rat microglial cells. Pyrimidinoceptors activate via a G protein the enzyme phospholipase C with the subsequent release of InsP3. The depletion of the intracellular Ca2+ pool appears to initiate a capacitative entry of Ca+ from the extracellular space. This Ca2+ then activates a Ca2+‐dependent K+ current.

Characterization and transduction mechanisms of purinoceptors in activated rat microglia.

1 Purinoceptor agonist‐induced currents in untreated (proliferating) and lipopolysaccharide‐ (LPS; 100 ng ml−1) treated (non‐proliferating) rat microglial cells were recorded by the whole‐cell patch‐clamp technique. 2 In non‐proliferating microglia, adenosine (0.01–100 μm), 2‐methylthio ATP (3–3000 nm), ATP (0.1–1000 μm), and ATP‐γ‐S (1–10 μm), but not α,β‐methylene ATP (α,β‐MeATP; 100 μm) produced a slow outward current at a holding potential of 0 mV. When K+ was replaced in the pipette solution by an equimolar concentration of Cs+ (150 mm), the 2‐methylthio ATP‐ (300 nm) induced outward current disappeared. The effect of 2‐methylthio ATP (300 nm) did not depend on the presence of extracellular Mg2+ (1 mm). The outward current response to 2‐methylthio ATP (300 nm) was larger in proliferating than in non‐proliferating microglia. 3 ATP (1–1000 μm) evoked a fast inward current at a holding potential of − 70 mV in non‐proliferating microglia, while adenosine (100–1000 μm) was inactive. When the effects of ATP were compared at 0 and − 70 mV, it became evident that ATP is much more potent in evoking the outward current. 4 The 2‐methylthio ATP‐ (300 nm) induced outward current was blocked by suramin (300 μm), but not by 8‐(p‐sulphophenyl)‐theophylline (100 μm), while the adenosine‐ (1 μm) induced outward current had the reverse sensitivity to these antagonists. 5 The 2‐methylthio ATP‐ (300 nm) induced outward current was inhibited by inclusion of GDP‐β‐S (200 μm) into the pipette solution or by preincubation of microglial cells with pertussis toxin (50 ng ml−1) for 12 h. The 2‐methylthio ATP‐ (300 μm) induced inward current was not changed by intracellular GDP‐β‐S (200 μm). The outward current response to adenosine (1 μm) was also abolished after pretreatment with pertussis toxin (50 ng ml−1). 6 Rat microglia possess both ATP‐sensitive P2Y‐ and adenosine‐sensitive P1‐purinoceptors. The ATP‐evoked inward current is mediated by P2Y‐purinoceptors, while the ATP‐ and adenosine‐evoked outward currents are mediated by P2Y‐ and P1‐purinoceptors, respectively. The transduction mechanisms of the outward, but not the inward current activation involve a pertussis toxin‐sensitive G protein.

ROLE OF ACTION POTENTIALS AND CALCIUM INFLUX IN ATP- AND UDP-INDUCED NORADRENALINE RELEASE FROM RAT CULTURED SYMPATHETIC NEURONES

Adenine and uracil nucleotides release noradrenaline from rat postganglionic sympathetic neurones by activation of P2X-receptors and distinct receptors for uracil nucleotides, respectively. The present study on cultured neurones of rat thoracolumbal paravertebral ganglia has analysed the involvement of action potentials and calcium influx in the nucleotide-induced transmitter release. ATP and UDP (100 µM each) caused a marked release of previously incorporated [3H]noradrenaline. The P2-receptor antagonists suramin (300 µM) and cibacron blue 3GA (3 µM) decreased the ATP-induced but not the UDP-induced release. The response to ATP was decreased by the sodium channel blocker tetrodotoxin (0.5 µM; by 47%), by the N-type calcium channel blocker ω-conotoxin GVIA (100 nM; by 35%), and by the α2-adrenoceptor agonist UK-14,304 (1 µM; by 45%); it was not changed by the potassium channel blocker tetraethylammonium (10 mM). The response to UDP was abolished by tetrodotoxin, greatly decreased by ω-conotoxin (by 78%), also abolished by UK-14,304, and increased by tetraethylammonium (by 410%). ATP (100 µM) caused a marked increase in intraaxonal free calcium as measured by fura-2 microfluorimetry. Withdrawal of extracellular calcium diminished the calcium response to ATP by 85%, and tetrodotoxin and ω-conotoxin diminished it by about 40%. As studied with the amphotericin B-perforated patch method, ATP (100 µM) induced a large depolarisation and action potential firing. UDP (100 µM) induced only a small depolarisation but it also elicited action potentials. UDP increased the excitability of the neurones. The results indicate that the ATP-induced release of noradrenaline depends on influx of calcium from the extracellular space. Calcium passes through two structures: voltage-gated channels and – probably – the P2X-receptor itself. Only that component of ATP-induced transmitter release which is triggered by opening of voltage-gated calcium channels is sensitive to modulation by α2-adrenocep-tors. In contrast to ATP, the UDP-induced release of noradrenaline is entirely due to generation of action potentials followed by calcium influx through voltage-gated channels. All of the UDP-induced release is therefore sensitive to α2-adrenoceptor modulation.

Inhibition of nicotinic acetylcholine receptor channels in bovine adrenal chromaffin cells by Y3-type neuropeptide Y receptors via the adenylate cyclase/protein kinase A system

The effect of neuropeptide Y [NPY(1–36)] and related peptides on the voltage-dependent currents and the nicotinic acetylcholine receptor (nAChR) currents (IACh) of bovine adrenal chromafptn cells was investigated using the whole-cell patch clamp technique. Catecholamine release from single chromaffin cells was measured by means of fast cyclic voltammetry. The potency order of these peptides in inhibiting IACh evoked by nicotine was NPY(1–36), NPY (16–36) > peptide YY(PYY) > [Leu31, Pro34] NPY. NPY(16–36) produced a similar degree of inhibition, irrespective of whether nicotine or an equipotent concentration of acetylcholine was used to evoke IACh. NPY(16–36) failed to alter voltage-dependent inward or outward currents. Intracellular cAMP, and extracellular dibutyryl-cAMP, produced a slowly developing increase in IACh. Intracellular cAMP, extracellular 8-Br-cAMP or dibutyryl-cAMP, and an inhibitor of cyclic nucleotide phosphodiesterases 3-isobutyl-l-methylxanthine (IBMX), decreased the inhibitory effect of NPY(16–36) on lACh. Although the intracellular application of the cAMP-dependent protein kinase A inhibitor [PKI(14–24)amide] alone did not alter IACh, it potentiated the effect of NPY(16–36) in interaction experiments. While the NPY(16–36)-induced inhibition of IACh was reversed on washout of the peptide, the slightly shorter C-terminal fragment NPY(18–36) caused a long-lasting depression of both IAch and catecholamine secretion evoked by nicotine. This depression was smaller in the presence of extracellular 8-Br-cAMP than in its absence. NPY(18–36) did not alter the secretory activity induced by a high concentration of potassium. It appears that, by activating Y3-receptors, NPY inhibits nAChR-current and the resulting secretion of catecholamines from bovine chromaffin cells. This process may involve a G protein-mediated decrease in intracellular cAMP with a subsequent decrease in the degree of phosphorylation of the nAChR-channel.

MODULATION OF LOCUS COERULEUS NEURONS BY EXTRA- AND INTRACELLULAR ADENOSINE 5'-TRIPHOSPHATE

The cell membrane of rat locus coeruleus (LC) neurons is sensitive to both extra- and intracellular ATP. Extracellular ATP or its enzymatically stable analogues activate membrane receptors of the P2 type. These receptors inhibit a persistent potassium current and simultaneously activate a nonselective cationic conductance. The resulting depolarization increases the spontaneous firing rate. A decrease in the concentration of intracellular ATP during hypoxia or hypoglycemia opens ATP-sensitive K+ (KATP) channels of LC neurons. The resulting hyperpolarization depresses the discharge of action potentials and conserved energy. The hypoxia-induced hyperpolarization is additionally due to the release of adenosine from neighboring neurons or glial cells. A certain class of compounds, termed potassium channel openers, also decrease the firing, while sulphonylurea antidiabetics known to block KATP channels increase it. Sulphonylurea antidiabetics antagonize the excitability decrease induced both by potassium channel openers and metabolic damage.