Tolbutamide attenuates diazoxide-induced aggravation of hypoxic cell injury

ATP-dependent potassium (K_ATP) channels of neurons are closed in the presence of physiological levels of intracellular ATP and open when ATP is depleted during hypoxia or metabolic damage. The present study investigates hypoxic alterations of purine and pyrimidine nucleotide levels supposed to intracellularly modulate K_ATP channels. In addition, the effects of the K_ATP channel activator diazoxide and its antagonist tolbutamide were investigated on ATP, GTP, CTP and UTP levels in slices of the parietal cortex. Hypoxia was evoked by saturation of the medium with 95% N₂–5% CO₂ instead of 95% O₂–5% CO₂ for 5 min. Nucleotide contents were measured by anion-exchange HPLC in neutralized perchloric acid extracts obtained from slices frozen immediately at the end of incubation. Hypoxia per se decreased purine and pyrimidine nucleoside triphosphate contents. Thus, ATP and GTP contents were reduced to 69.9 and 77.6% of the respective normoxic levels. UTP and CTP contents were even more decreased (to 60.9 and 41.6%), probably because the salvage pathway of these pyrimidine nucleotides is less effective than that of the purine nucleotides ATP and GTP. While tolbutamide (300 μM) had no effect on the hypoxia-induced decrease of nucleotides, diazoxide at 300, but not 30 μM aggravated the decline of ATP, UTP and CTP to 51.8, 37.5 and 28.5% of the contents observed at normoxia; GTP levels also showed a tendency to decrease after diazoxide application. Tolbutamide (300 μM) antagonized the effects of diazoxide (300 μM). Nucleoside diphosphate (ADP, GDP and UDP) levels were uniformly increased by hypoxia. There was no hypoxia-induced increase of ADP contents in the presence of tolbutamide (300 μM). The ATP/ADP, GTP/GDP and UTP/UDP ratios uniformly declined at a low pO₂. However, only the ATP/ADP ratio was decreased further by diazoxide (300 μM). The observed alterations in nucleotide contents may be of importance for long- and short-term processes related to acute cerebral hypoxia. Thus, hypoxia-induced alterations of purine and pyrimidine nucleotide levels may influence the open state of K_ATP-channels during the period of reversible hypoxic cerebral injury. Furthermore, alterations during the irreversible period of cerebral injury may also arise, as a consequence of decreased pyrimidine nucleotide contents affecting cell survival via protein and DNA synthesis.     

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.     

Effects of divalent cations and normorphine on spontaneous excitatory junction potentials in the mouse vas deferens.

1 Excitatory junction potentials (e.j.ps) occurring spontaneously or evoked by nerve stimulation were recorded intracellularly from smooth muscle cells of the mouse isolated vas deferens. 2 The amplitude of the evoked e.j.ps and the amplitude and frequency of spontaneous e.j.ps were measured before and during application of normorphine or solutions which might be expected change the influx of calcium ions into the nerve terminals. 3 Spontaneous e.j.ps could be recorded even in solutins which contained tetrodotoxin (1 microM), no added calcium an EGTA (1mM). A four fold increase in calcium concentration from 1.25 to 5 mM greatly increased the amplitude of the evoked e.j.ps but had no effect on the amplitude or frequency of the spontaneous e.j.ps. 4 Magnesium (12mM) and cobalt (4mM) both greatly reduced the evoked e.j.ps and also reduced the frequency of spontaneous e.j.ps. 5 Normorphine (2 microM) reduced the amplitude of the evoked e.j.p by 70% but had no effect on the amplitude or frequency of spontaneous e.j.ps. 6 It is suggested that normorphine inhibits noradrenaline secretion from nerve varicosities by a mechanism different from that of magnesium and cobalt. One possibility is a block of action potential propagation along varicose fibers.     

4-Aminopyridine and evoked transmitter release from motor nerve endings

1 In the presence of tetrodotoxin, electrotonic depolarization of frog motor nerve terminals causes the appearance of stimulus-graded endplate potentials. When 4-aminopyridine is added, the graded endplate potential is converted into a triggered all-or-none response resulting in giant endplate potentials of about 70 mV amplitude and 50 ms duration. The triggered endplate potentials are abolished in Ca²⁺-free saline and are blocked by Mn²⁺ ions. Sr²⁺ but not Ba²⁺ can replace Ca²⁺ in supporting transmitter release. Mg²⁺ fails, even in concentrations as high as 32 mM, to affect the amplitude and the shape of the endplate potential but abolishes it when the Ca²⁺ concentration is reduced to 0.2 mM.

2 Despite the large amplitude of the triggered endplate potential in the presence of 4-aminopyridine and tetrodotoxin, repetitive stimulation up to 10 Hz causes only a small decline in amplitude of successive endplate potentials. However, in the presence of (+)-tubocurarine or gallamine, repetitive nerve stimulation produces a marked decline in successive endplate potential amplitude. The fall is counteracted when evoked transmitter release is reduced in the presence of 0.2 mM Ca²⁺. The results suggest that in the presence of 4-aminopyridine such large amounts of transmitter are released that even during repetitive stimulation (5 to 10 Hz) endplate potentials are of maximal amplitude.

3 4-Aminopyridine causes a prallel shift to the right of the dose-response curve to Mg²⁺ for blockade of nerve impulse-evoked transmitter release (in the absence of tetrodotoxin). A similar parallel shift occurs in the presence of tetraethylammonium and guanidine.

4 It is concluded that 4-aminopyridine increases transmitter release by enhancing the transport efficacy for Ca²⁺ across the nerve terminal membrane during nerve terminal depolarization.

     

P2 receptor-stimulation influences axonal outgrowth in the developing hippocampus in vitro

Extracellular ATP might act as a trophic factor on growing axons during development of the CNS via P2 receptors. In the present study the postnatal presence of selected P2 receptor subtypes was analyzed and their putative trophic capacity in entorhino-hippocampal slice co-cultures of mouse brain was tested. The effect of the P2 receptor ligands 2-methylthioadenosine-5'-triphosphate (P2X/Y receptor agonist) and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (P2X/Y receptor antagonist) on axonal growth and fiber density of biocytin-labeled hippocampal projections was compared both with untreated cultures and with cultures treated with artificial cerebrospinal fluid. After 10 days in vitro, double immunofluorescence labeling revealed the expression of P2X(1), P2X(2), P2X(4) as well as P2Y(1) and P2Y(2) receptors in the examined regions of entorhinal fiber termination. Further, quantitative analysis of identified biocytin-traced entorhinal fibers showed a significant increase in fiber density in the dentate gyrus after incubation of the slices with the P2 receptor agonist 2-methylthioadenosine-5'-triphosphate. This neurite outgrowth promoting effect was completely abolished by the P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid. Our in vitro data indicate that ATP via its P2X and P2Y receptors can shape hippocampal connectivity during development.     

Co-transmitter function of ATP in central catecholaminergic neurons of the rat

Intracellular recordings were made in a mid-pontine slice preparation of the rat brain containing the nucleus locus coeruleus. Focal electrical stimulation evoked biphasic synaptic potentials consisting of early depolarizing (d.p.s.p.) and late hyperpolarizing (i.p.s.p.) components. The alpha(2)-adrenoceptor antagonist idazoxan inhibited the i.p.s.p. without altering the d.p.s.p. All of the following experiments were carried out in the presence of kynurenic acid and picrotoxin to block the glutamatergic and GABAergic fractions of the d.p.s.p., respectively. Guanethidine, which is known to inhibit noradrenaline and ATP release from nerve terminals of postganglionic sympathetic nerves, depressed both the d.p.s.p. and the i.p.s.p. in a concentration-dependent manner. Damage of catecholaminergic nerve terminals by 6-hydroxydopamine also decreased both the d.p.s.p. and the i.p.s.p. The P2 receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) depressed the d.p.s.p., whereas the i.p.s.p. remained unaffected. The further application of PPADS did not increase the depression of the d.p.s.p. by guanethidine. Superfusion with the mixed alpha-adrenoceptor agonist noradrenaline or the selective P2 receptor agonist adenosine 5'-O-(2-thiodiphosphate) inhibited both the d.p.s.p. and the i.p.s.p. The inhibitory effects of these agonists were prevented by the respective antagonists idazoxan or suramin. In the presence of suramin noradrenaline failed to inhibit the residual d.p.s.p. Superfused noradrenaline potentiated rather than inhibited responses to pressure-applied alpha,beta-methylene-ATP; superfused adenosine 5'-O-(2-thiodiphosphate) did not interact with pressure-applied noradrenaline. In conclusion, we present electrophysiological evidence for the co-release of ATP and catecholamines in the CNS. At the cell somata of neurons in the locus coeruleus, noradrenaline and ATP activate inhibitory alpha(2)-adrenoceptors and excitatory P2 receptors, respectively. In addition, inhibitory presynaptic autoreceptors of the alpha(2) and P2 types appear to regulate release of the two co-transmitters.     

Loudness dependence of auditory evoked potentials (LDAEP) in clinical monitoring of suicidal patients with major depression: a pilot study

Loudness dependence of auditory evoked potentials (LDAEP) is a validated in vivo marker of central serotonergic function. We aimed at measuring serotonergic activity in a follow-up study of suicidal patients. It should be investigated whether suicide attempts or suicidal states cause changes in the LDAEP. Thirteen patients (mean age, 40.9?±?11.3?years; age range, 20-61, 6 male) with a major depressive episode who had attempted suicide or had suicidal plans (Hamilton Depression Rating Scale item 3 [suicidality] ≥3) were included in the study. LDAEP and psychometric measurements took place about 2, 5, 9 and 16?days after attempted suicide or suicidal action. On day 9, LDAEP was significantly higher compared to day 2 and day 16; there was a similar tendency compared to day 5. Instability of central serotonergic function is suggested resulting in reduced serotonergic activity about 1?week after suicide attempt. Further studies are necessary that include larger samples in order to distinguish between different psychiatric diseases and to consider confounding factors like gender, smoking, medication, impulsivity or lethality of suicidal action.