Margit Dóda

Regulatory interactions among axon terminals affecting the release of different transmitters from rat striatal slices under hypoxic and hypoglycemic conditions

Abstract: An in vitro model of ischemia was utilized to study the effects of both oxygen and glucose depletion on transmitter release from rat striatal slices. The spontaneous and stimulation‐evoked releases of tritiated dopamine, γ‐aminobutyric acid, glutamate, and acetylcholine were measured. Hypoxia increased the evoked release of glutamate and dopamine without effect on the resting release. In contrast, hypoglycemia itself increased the resting release of dopamine. Hypoxia in combination with hypoglycemia provoked a massive release of glutamate, dopamine, and γ‐aminobutyric acid. The effect on acetylcholine release was less pronounced. Ca2+ withdrawal partly reduced the effect of hypoxia combined with hypoglycemia on dopamine release and application of tetrodotoxin (1 μM) abolished it. MK‐801 (3 μM), an N‐methyl‐d‐aspartate receptor antagonist, attenuated the effect of hypoxia and hypoglycemia on [3H]dopamine release. ω‐Conotoxin (0.1 μM) had a similar effect on stimulation‐evoked release under a hypoxic condition. The D2 receptor antagonist sulpiride (100 μM) failed to enhance the release of [3H]acetylcholine in hypoxia combined with hypoglycemia. It was suggested that in response to hypoxia combined with hypoglycemia there is a massive release of glutamate due to the increased firing rate which in turn releases dopamine from the axon terminals through stimulation of presynaptic N‐methyl‐d‐aspartate receptors. Dopaminergic inhibitory control on ACh release seems not to be operative under conditions of hypoxia combined with hypoglycemia.

Ischemic-like condition releases norepinephrine and purines from different sources in superfused rat spleen strips

Transmitters and cotransmitters of the sympathetic nervous system are involved in the regulation of a variety of immune cell functions. However, it is not entirely clear what stimuli lead to the release of these molecules in immune organs. In this study, we investigated whether local ischemia can cause the parallel release of norepinephrine and its cotransmitter, ATP, in the spleen. Ischemic-like conditions, simulated by transient (15 min) O(2) and glucose deprivation, elicited a reversible increase in the release of both norepinephrine and purines from superfused spleen strips preloaded with [3H]norepinephrine or [3H]adenosine. HPLC analysis of the released tritium label revealed a net increase in the amount of ATP, ADP, AMP, adenosine, inosine, hypoxanthine and xanthine in response to ischemic-like condition. Selective O(2) or glucose deprivation, and Ca(2+)-free conditions differentially affected the outflow of [3H]norepinephrine and [3H]purines, indicating that they derived from different sources. The ABC transporter inhibitors glibenclamide (100 microM) and verapamil (100 microM) as well as low-temperature inhibited [3H]purine release evoked by ischemic-like conditions. Surgical denervation of the spleen reduced endogenous catecholamine content and [3H]norepinephrine uptake of the spleen, but not that of [3H]adenosine. In summary, these results demonstrate the release of norepinephrine and purines in response to an ischemic-like condition in an immune organ. Although both could provide an important source of extracellular catecholamines and purines involved at various levels of immunomodulation, the source and mechanism of norepinephrine and purine efflux seem different.

Functional evidence that acetylcholine release from Auerbach's plexus of guinea-pig ileum is modulated by α2A-adrenoceptor subtype

The effects of alpha 2-adrenoceptor ligands on electrically stimulated [3H]acetylcholine release from longitudinal muscle strips of guinea-pig ileum were examined. Xylazine or oxymetazoline reduced the release of [3H]acetylcholine in a concentration-dependent manner, both these actions being antagonized by idazoxan, CH 38083, or WB 4101. Prazosin, considered as an alpha 2B-adrenoceptor antagonist, failed to modify the inhibitory effects of xylazine or oxymetazoline. It is concluded that the alpha 2-adrenoceptors involved in the modulation of acetylcholine release from cholinergic axon terminals of guinea-pig ileum are of the alpha 2A subtype.

Coloured peptides: synthesis, properties and use in preparation of peptide sub-library kits.

Several methods were developed for the solid‐phase synthesis (SPPS) of coloured peptides and peptide libraries. At first a bifunctional red compound, 4‐(4‐(N‐ethyl‐N‐(3‐(tert‐butyloxycarbonyl)aminopropyl)amino)phenylazo)benzoic acid (Boc‐EPAB), was coupled with chloromethyl resin to obtain a new solid support suitable for SPPS using Boc chemistry. Peptides synthesized on this coloured resin had the chromophore at their C‐termini. N‐terminally coloured peptides were synthesized on a traditional solid support, coupled with chromophoric carboxylic acid before cleavage. A model pentapeptide, Phe‐Ala‐Val‐Leu‐Gly, and its ten derivatives were synthesized and their properties studied. It was found that the presence of chromophores decreases the water solubility of peptides. However, insertion of solubilizing tags (penta‐lysine sequences or polyoxyethyl chains) into the molecule of any coloured derivative resulted in enhancement of the solubility. The RP‐HPLC hydrophobicity indexes (φ0) of the coloured peptides were also determined because φ0 values are closely related to their water solubility. A coloured pentapeptide library was synthesized using the portioning‐mixing method. Each component of this library contained the red azo dye (EPAB) and the penta‐lysine tag. Before the last coupling step the samples were not mixed. All of the 19 sub‐libraries obtained after cleavage were readily soluble in water, giving intense red solutions.

Role of Different Subtypes of Adrenoceptors in Pressor Responses to Catecholamines Released From Sympathetic Nerve Endings

The role of alpha 1- and alpha 2-adrenoceptors in the vascular effects of catecholamines, either released locally from sympathetic nerve endings (e.g., in vascular smooth muscle) or derived from the adrenal medulla or administered intravenously, was studied using selective antagonists of these adrenoceptors. The ganglionic stimulant dimethylphenyl-piperazinium-iodide (DMPP) exerted dual actions on blood pressure: a rapid and short-term pressor reaction (phase I) resulting from catecholamine release elicited by ganglion stimulation, followed by a more sustained blood pressure elevation (phase II) resulting from the circulating catecholamines released from the adrenal medulla. The selective alpha 2-adrenoceptor, but a not subtype selective, antagonist 7,8-(methylenedioxi)-14-alpha-alloberbane HCl (CH-38083) (50-100 micrograms/kg, IV) significantly (p < 0.05) inhibited the pressor effects of epinephrine and norepinephrine given intravenously and phase II of the DMPP-induced pressor reaction. Idazoxan exerted similar effects, but at higher doses (400-600 micrograms/kg, IV). WB-4101 (50-100 micrograms/kg, IV) and BRL-44408 (2-3 mg/kg, IV), two selective alpha 2A-adrenoceptor antagonists, had the same activity as CH-38083, except did not inhibit the pressor effect of intravenously administered norepinephrine. The alpha 2B-adrenoceptor selective antagonist, ARC-239 (150 micrograms/kg, IV) did not influence phase II of DMPP-induced pressor reaction. Prazosin (200 micrograms/kg, IV), an antagonist of alpha 1 and alpha 2B-adrenoceptors, reduced blood pressure, the pressor response to intravenously administered epinephrine, and phase I of the DMPP-induced pressor effect. In addition, it completely inhibited the pressor responses to DMPP remaining after administration of CH-38083. These results suggest that the postsynaptically located alpha 1- and alpha 2(A and B)-adrenoceptors are involved in pressor response to norepinephrine and epinephrine, and are sensitive and accessible to catecholamines released locally from the axon terminals, and from the circulation to a different extent. These results may have great therapeutical importance in hypertension, for which the involvement of both a high level of circulating and locally released catecholamines may be indicative of the usefullness of a combination (alpha 1- and alpha 2-adrenoceptors- and Ca-channel-blocking agents) therapy.

Fluorescent derivatives of bovine neurotensin 8-13 fragment

Fluorescent derivatives of bovine neurotensin 8–13 fragment were prepared. For N-terminal labelling, 4-[7-hydroxycoumaryl]acetic acid (Hca), 4-[7-methoxycoumaryl]acetic acid (Mca) and 2-amino-3-[4-[7-methoxycoumaryl]]propionic acid (Amp) were used while the C-terminus of the peptide chain was elongated with Amp. The fluorescence excitation and emission spectra of the peptide derivatives were studied. Hca- and Mca/Amp-derivatives were easily distinguishable because of the 60 nm shift of their emission maxima. Compared with the natural sequence, the presence of an N-terminal label did not influence the biological potency in a longitudinal muscle strip of guinea-pig ileum, while labelling at the C-terminus considerably reduced the activity of the peptide.

Effect of CH-38083, a selective antagonist of alpha2-adrenoceptors, on renal sympathetic function

The effects of 7,8-(methylenedioxi)-14-alpha-hydroxyalloberbane HCl (CH-38083), a structurally new, selective and potent alpha 2-adrenoceptor antagonist, and of idazoxan, were studied on both the spontaneous activity of the postganglionic sympathetic renal nerve and on the clonidine- or xylazine-induced inhibitory action in anaesthetized cats. The drug CH-38083 (30-200 micrograms/kg, i.v.) caused a sustained increase of the sympathetic activity and blood pressure. Larger doses of idazoxan (200-500 micrograms/kg, i.v.) were needed to induce similar effects. Both antagonists were effective in antagonizing the sympatho-inhibitory effect of clonidine or xylazine. The excitatory response to selective alpha 2-adrenoceptor antagonists suggests that the sympathetic output undergoes tonic inhibition due to a permanent alpha 2-adrenoceptor stimulation.

Central cholinergic interactions in somato-vegetative reflexes

Abstract In cats lightly anaesthetized with urethane (600 mg/kg, i.p.), electrical stimulation of the sciatic nerve elicited frequency-dependent pressor reflexes and contractions of the nictitating membrane. Administration of oxotremorine (0.2 mg/kg, i.v.) or physostigmine (0.5 mg/kg, i.p.) resulted in depression of the pressor reflexes. At the same time, physostigmine enhanced the reflex contractions of the nictitating membrane, while oxotremorine induced sustained contraction of the latter. All these effects were antagonized by the tertiary amine scopolamine, but not by the quaternary atropine methylbromi. The results point to a role of central cholinergic mechanisms in the integration of somato-vegetative reflexes, and give evidence that the sympathetic driving of different effectors is not uniformly organized by the central nervous system.

Oxotremorine: acute tolerence to it and its central “cholinolytic” effect in mice

Decsi, VSlrszegi & MChes (1961a, b) reported that tremorine lost much of its analgesic, tremorigenic and narcosis-potentiating properties when given to mice. Keranen, Zaratzian & Coleman (1961) observed a progressive decline of the compound’s tremorigenic effect after chronic treatment of unstated duration. Oelszner (1965) was unable to corroborate the observation on the analgesic effect of tremorine made by Decsi & others. Doses of tremorine and oxotremorine prevent albino mice from clinging for more than 3 to 6 s to a rod operated at 7 rotations/min. Activity was regarded as negative in experiments in which the animal held fast to the rotarod for at least 180 s. The doses given are for oxotremorine oxalate, physostigmine salicylate, nicotine hydrogen tartrate, and tremorine dichlorhydrate. Table 1 shows that a first injection of tremorine reduces the rotarod activity of a second injection given 16 h later, and totally prevents that of oxotremorine. The rotarod activity of oxotremorine (0.5-1.0 mg/kg, i.p.) wears off within 60-90 min.

Effect of bimoclomol (N-[2-hydroxy-3-(1-piperidinyl) propoxy]-3 pyridine-carboximidoyl-chloride) on iminodipropionitrile-induced central effects

Dyskinesia is frequently seen in neurological disorders affecting the basal ganglia. Iminodipropionitrile (IDPN) produces a somewhat similar motor syndrome in rodents, one that is a possible model for dyskinesia. Because in previous studies the compound (N-[2-hydroxy-3-(1-piperidinyl) propoxy]-3 pyridine-carboximidoyl-chloride) (Bimoclomol, BRLP-42) was shown to provide protection against IDPN-induced retinopathy; we investigated the effect of BRLP-42 on IDPN-induced motor changes and on IDPN-induced cerebral amino acid level changes in rats and mice. IDPN had a biphasic effect on motor activity in C57BL/6 mice: it was a depressant for 24 days and a stimulant after 30 days. Bimoclomol inhibited the motor depressant effect and enhanced the stimulatory effect of IDPN in this mouse strain. In BALB/cBy mice and Sprague Dawley rats IDPN produced persistent vertical head movements and changes in the level of glutamic acid in brain. Bimoclomol reduced the effect of IDPN on head movements and blocked the effect on cerebral glutamate; by itself it had no effect on motor activity in either species. Bimoclomol inhibited ischemia-induced [3H]norepinephrine release from rat hippocampal slices. Our findings indicate that Bimoclomol could have a beneficial effect on some dyskinesias, and on drug-induced vertical head movements.