Peter Illes

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.

Identification of the neuroeffector transmitter in jejunal branches of the rabbit mesenteric artery

SummaryVasoconstriction or excitatory junction potentials (e.j.ps) evoked by nerve stimulation (15 field pulses at 2 Hz every 3 min) were recorded in rabbit isolated jejunal arteries. The resting diameter of the arteries and its decrease in response to stimulation was measured by a photoelectric method. Vasoconstriction was insensitive to prazosin 0.1 or 1 μmol/l. Yohimbine 1 μmol/l considerably enhanced, whereas α,β-methylene ATP (α,β-meATP) 1 μmol/l abolished the contractile response. In order to test the effect of exogenously applied transmitter candidates, noradrenaline (0.1–1 μmol/l) and ATP (10–30 μmol/l) were added in concentrations which evoked a vasoconstriction comparable to that induced by electrical stimulation. The action of noradrenaline was prevented by prazosin 0.1 μmol/l, but was unaffected by both yohimbine 1 μmol/l and α,β-meATP 1 μmol/l. α,β-meATP 1 μmol/l depressed the effect of ATP. The e.j.ps evoked by a train of 15 pulses showed facilitation up to the third response and thereafter depression; a partial summation was also observed. Prazosin 0.1 μmol/l did not change the e j.p. amplitudes. By contrast, when yohimbine 0.1 or 1 μmol/l was added to the prazosin-containing medium, both the late e j.ps in the train and the summation were enhanced in a concentration-dependent manner. α,β-meATP 1 μmol/l almost abolished the e.j.ps. In conclusion, in rabbit jejunal arteries, stimulation of postganglionic sympathetic nerves may release noradrenaline together with ATP which is probably the sole neuroeffector transmitter under our conditions. Transmitter release seems to be modulated by the activation of presynaptic α2-adrenoceptors. Under the stimulation conditions of the present experiments the released transmitter does not activate postsynaptic α1-adrenoceptors.

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.

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 P2-purinoceptor agonist alpha,beta-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, alpha,beta-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 alpha,beta-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 alpha,beta-methylene ATP effect. In the hyperpolarized region, the current-voltage curve of alpha,beta-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 alpha,beta-methylene ATP; external tetraethylammonium was also inhibitory. External Ba2+ and Cs+ had no effect or only slightly decreased the alpha,beta-methylene ATP-induced depolarization. A low Na+, or a low Ca2+ high Mg2+ medium, as well as the presence of Co2+ in the medium, markedly reduced or even abolished the depolarization by alpha,beta-methylene ATP. ATP itself did not produce consistent changes in the membrane potential or input resistance. However, in the presence of the P1-purinoceptor antagonist 8-cyclopentyl-1,3-dipropylxanthine, ATP consistently increased the firing rate and evoked an inward current. In conclusion, P2-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.

Galanin receptors inhibit the spontaneous firing of locus coeruleus neurones and interact with μ-opioid receptors

Electrophysiological experiments were performed in a pontine slice preparation of rat brain containing the locus coeruleus (LC). The extracellular part of this study showed that galanin (0.003-0.1 mumol/l), [Met5]enkephalin (0.01-10 mumol/l) and noradrenaline (0.1-100 mumol/l) concentration dependently inhibited the firing rate. Noradrenaline (1 and 3 mumol/l) had the same effect both before and during the application of galanin (0.001 or 0.01 mumol/l). Similarly, [Met5]enkephalin (0.03 and 0.1 mumol/l) produced identical inhibition, regardless of the presence or absence of 0.01 mumol/l galanin. Whereas rauwolscine (1 mumol/l) potentiated the effect of galanin (0.03 mumol/l), idazoxan (1 mumol/l) was inactive. In contrast, both naloxone (0.1 mumol/l) and beta-funaltrexamine (0.1 mumol/l) facilitated the galanin-induced inhibition. In the intracellular experiments, galanin (0.3 mumol/l) abolished the spontaneous discharge of action potentials, hyperpolarized the cells and decreased their input resistance. In conclusion, galanin may depress the firing rate by increasing a potassium permeability. Moreover, galanin receptors appear to interact with mu-opioid receptors but not with alpha 2-adrenoceptors.

Inhibition of noradrenaline release by ω-conotoxin GVIA in the rat tail artery

1 The perivascular nerves of isolated tail arteries from Wistar rats were stimulated with field pulses (1Hz, 2 pulses, every 2 min). ω‐Conotoxin 10 nmol l−1 depressed neurogenically mediated contractions, but did not influence the contractions to noradrenaline 0.1–0.3 μmol 1−1. 2 The inhibitory effect of ω‐conotoxin was concentration‐dependent (IC50 = 3.8 nmol l−1). It did not reach a steady‐state during 30 min incubation and could not be reversed upon subsequent washout for another 60 min. 3 A gradual increase in the Ca2+ concentration of the medium from 1.25 mmol l−1 to 10 mmol 1−1 enhanced vasoconstriction and attenuated the action of ω‐conotoxin 10 nmol l−1. When a low stimulation intensity (120 mA) was used at high external Ca2+ (10 mmol l−1), similar contractile responses were obtained as under normal conditions (200mA current, 2.5 mmol l−1 Ca2+). However, the inverse relationship between the effect of the toxin and external Ca2+ remained unchanged. 4 The time‐course and degree of the inhibition by ω‐conotoxin 3 nmol l−1 was identical in tail arteries of spontaneously hypertensive rats (SHR) and their normotensive controls (WKY). 5 When tail arteries of Wistar rats were preincubated with [3H]‐noradrenaline, field stimulation (0.4 Hz, 24 pulses, every 16 min) evoked tritium overflow and vasoconstriction. ω‐Conotoxin 30 nmol l−1 inhibited both responses to a similar extent. 6 Our results suggest that ω‐conotoxin selectively blocks Ca2+ channels in the terminals of perivascular nerves and thereby reduces the release, but not the contractile effect of the sympathetic transmitter.

Characterization of Opioid Receptors Modulating Noradrenaline Release in the Hippocampus of the Rabbit

Noradrenaline (NA) release and its modulation via presynaptic opioid receptors were studied in rabbit hippocampal slices, which were preincubated with [3H]NA, continuously superfused in the presence of 30 μM cocaine and stimulated electrically. The evoked release of [3H]NA was strongly reduced by the preferential K‐agonists ethylketocyclazocine, dynorphin A1‐13, dynorphin A, trans‐3,4‐dichloro‐N‐methyl‐N‐[2‐(1‐pyrrolidinyl)‐cyclohexyl]‐benzeneacetamide (U‐50,488), and (–)‐5,9‐dimethyl‐2′‐OH‐2‐tetrahydrofurfuryl‐6,7‐benzomorphan [(–)‐MR 2034], whereas (+)‐MR 2035 [the (+)‐enantiomer of (–)‐MR 2034] was ineffective. In contrast, the preferential δ‐agonists Leu‐enkephalin, Met‐enkephalin, and D‐Ala2,‐D‐Leu5‐enkephalin (DADLE) as well as the μ‐agonists morphine, normorphine, D‐Ala2Gly‐ol5‐enkephalin (DAGO), and β‐casomorphin1‐4 amide (morphiceptin) were much less potent. However, in similar experiments on rat hippocampal slices DAGO (1 μM) was much more potent than ethylketocyclazocine (1 μM) or DADLE (1 μM). (–)‐N‐(3‐furylmethyl)‐α‐noretazocine [(–)‐MR 2266], 1 μM, a preferential K‐antagonist, antagonized the effect of ethylketocyclazocine more potently than (–)‐naloxone or (+)‐MR 2267 [the (+)‐enantiomer of (–)‐MR 2266]. Given alone, (–)‐MR 2266 slightly and (+)‐MR 2267 (1 μM each) greatly enhanced NA release, apparently due to α2‐adrenoceptor blockade since their effects were completely abolished in the presence of yohimbine (0.1 μM). The effects of DADLE (1 μM) and DAGO (1 μM) were also antagonized by (–)MR 2266 (0.1 μM) but not by the δ‐antagonist N,N‐diallyl‐Tyr‐Aib‐Phe‐Leu‐OH (ICI 174864), 0.3 μM. It is concluded that NA release in the rabbit hippocampus is inhibited via K‐receptors; our results do not support the presence of modulatory μ‐ and δ‐receptors in this tissue. However, in the rat hippocampus μ‐receptors may modulate NA release.

Hypoxic changes in rat locus coeruleus neurons in vitro.

1. Intracellular recordings were made in a pontine slice preparation of the rat brain containing the nucleus locus coeruleus (LC). Locus coeruleus neurons responded to brief hypoxic stimuli (replacement of 95% O2‐5% CO2 with 95% N2‐5% CO2) with hyperpolarization and a cessation of spontaneous action potentials. When the cells were continuously hyperpolarized by about 15 mV in order to abolish spontaneous firing, hypoxia induced an early depolarization (HD), followed by a hypoxic hyperpolarization (HH) and after reoxygenation, a posthypoxic hyperpolarization (PHH). These responses were accompanied by a decrease in input resistance, which was larger during HH than during HD but, thereafter, became smaller during PHH. 2. The hypoxia‐induced currents associated with the changes in membrane potential, at a holding potential of ‐70 mV, were an early inward current (HIC), a subsequent outward current (HOC) and after reoxygenation, another outward current (PHOC). The HIC did not change with an increasing holding potential. In contrast, the HOC reversed its amplitude at about ‐95 mV. Finally, the PHOC decreased, but did not reverse its polarity at more negative holding potentials. When the external K+ was elevated from 2.5 to 10.5 mM, the current‐voltage (I‐V) relation of the HOC and its reversal potential were shifted to the right. 3. In the presence of tetrodotoxin, the HH decreased. A low Ca(2+)‐high Mg2+ medium depressed both the HH and PHH. Rauwolscine did not alter either response to hypoxia, while 8‐cyclopentyl‐1,3‐dipropylxanthine decreased the PHH only. S‐(p‐Nitrobenzyl)‐6‐thioguanosine potentiated both HH and PHH. 4. Whereas tolbutamide markedly lowered the HH and PHH, glibenclamide was ineffective. Tetraethylammonium also failed to alter the hypoxic responses. Furthermore, ouabain or the removal of K+ from the superfusion medium, depressed PHH. 5. Pressure application of adenosine inhibited the spontaneous firing of LC neurons. DPCPX did not alter the firing, but antagonized the effect of adenosine. Tolbutamide also counteracted the inhibitory effect of adenosine and, additionally, facilitated the firing rate in some neurons. Moreover, tolbutamide abolished the adenosine‐induced outward current. 6. Early hypoxic depolarization and PHH are mostly due to the blockade and subsequent reactivation of the K(+)‐Na+ pump, respectively. The HH is caused by the opening of ATP‐sensitive K+ (KATP) channels in response to the hypoxia‐induced decline of intracellular ATP. Adenosine released by hypoxic stimuli may lead to an adenosine A1‐receptor‐mediated opening of (KATP) channels during the HH and more markedly during the PHH.