The nonspecific P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), the nonspecific P1 receptor antagonist 8-(p-sulphophenyl)-theophylline (8-SPT) and the combination of both were applied by retrograde microdialysis into the nucleus accumbens (NAc) before and during feeding of 18-h food-deprived rats. In addition to the registration of behavioural parameters, such as the amount and duration of food intake, the feeding-induced changes in dopamine (DA) concentration and the concomitant changes of neuronal activity in the NAc and the ventral tegmental area (VTA) were simultaneously determined. The perfusion with PPADS (20 mum) diminished the amount of food intake and the duration of feeding. Furthermore, the P2 receptor antagonist blocked the feeding-induced DA release and prevented the feeding-elicited changes of the electroencephalography (EEG) power distribution which was characterised by an increase in the power of the 8.0-13.0-Hz frequency band in the NAc and the VTA. The effects of PPADS could be completely prevented by the concomitantly perfused adenosine receptor antagonist 8-SPT (100 mum). When given alone, 8-SPT increased the amount of food ingested, the duration of feeding and the EEG power of the higher frequency range, particularly between 19.0 and 30.0 Hz, in both the NAc and the VTA. The feeding-elicited DA release was supplemented to the enhanced DA level caused by the perfusion with 8-SPT in an additive manner. The P2 and P1 receptor antagonists interact antagonistically in the modulation of feeding behaviour and the feeding-induced changes of EEG activity suggesting that both endogenous extracellular ATP and adenosine are involved in the regulation of the feeding-associated mesolimbic neuronal activity in a functionally antagonistic manner.

The effects of the P2 receptor ligands 2-methylthio ATP (2-MeSATP; 10 pmol)--as a non-specific agonist--and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; 10 pmol)--as a non-selective antagonist--after bilateral intra-accumbens injection on the locomotor response were investigated in an open field situation. The P2 receptor-mediated effects on the pattern of locomotor activity were compared with the effects caused by the dopamine D2-like receptor agonist quinpirole (10 pmol) and by the combination of the N-methyl-D-aspartate (NMDA) receptor antagonist (+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP; 10 pmol) with the alpha-amino-3-hydro-5-methyl-4-isoxazolpropionic acid (AMPA) and kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 30 pmol). The intra-accumbens injection of all tested compounds elicited an increase in the locomotor activity over a test period of 20 min when compared with the controls. No statistically significant differences could be evaluated between the different drug-treated groups. However, a more detailed analysis--using further behavioural parameters such as the number of movement direction changes, the effective running time and the running speed--revealed two basically different patterns of locomotor activity. The locomotor response induced by the injection of 2-MeSATP or quinpirole was characterised by a continuous and consistent locomotion, whereas the enhanced locomotor activity elicited by PPADS or CPP/CNQX was determined by an increased running speed accompanied by more disruptions and more changes of movement direction. The coadministration of 2-MeSATP and quinpirole led to an enhancement of locomotor activity in a limited post-treatment interval. The effects of both compounds could be abolished by the pre-treatment with the D2/D3 receptor antagonist sulpiride (100 pmol). Coadministration of PPADS and CPP/CNQX caused additive effects suggesting that the pathway mediated by P2 and ionotrophic glutamate receptors is different. The stimulation of P2 receptors in the nucleus accumbens (NAc) modulates the locomotion in the direction to be to be longer lasting, more consistent and more goal directed.

Rationale: Previous experiments have shown that P2 receptor activation increases the release of dopamine in the mesolimbic mesocortical system. Objective: In order to investigate the functional correlates of dopaminergic stimulation, EEG and behavioural responses to injection of the P2 receptor agonist 2-methylthio ATP (2-MeSATP) into the nucleus accumbens (NAc) of rats were investigated. Methods: EEG electrodes were positioned into the NAc together with the guide cannula for intracerebral injection. Behavioural analysis was performed in an open field cage and was evaluated by a video activity measurement system. Rats were assigned to separate groups that were given artificial cerebrospinal fluid (aCSF) or drug treatment. Results: 2-MeSATP significantly extended the period of locomotor activity in the novel environment. The quantitative EEG was characterized by an elevation of the power in the alpha-1 range and a decrease in power in the delta range. The P2 receptor antagonists reactive blue 2 but not pyridoxalphosphate-6-azophenyl-2'4'-disulphonic acid (PPADS) also enhanced locomotion when given alone, and elevated the alpha-1 and beta-2 bands. Both antagonists abolished the locomotor and EEG responses to 2-MeSATP. The dopamine D1 receptor antagonist SCH 23390 and the D2/D3 receptor antagonist sulpiride did not alter locomotor activity when given either alone or in combination. Only sulpiride and especially sulpiride in combination with SCH 23390 prevented the effect of 2-MeSATP. Sulpiride produced a selective increase in the alpha-1 band of the power spectrum whereas SCH 23390 elevated the power of the alpha-1, alpha-2 and beta-1 activities. Neither antagonist inhibited the effect of 2-MeSATP on the EEG when applied separately; however, the co-administration of SCH 23390 and sulpiride abolished the 2-MeSATP-induced alteration of power distribution. After a 6-hydroxydopamine (6-OHDA)-induced lesion of the accumbal dopaminergic terminals, 2-MeSATP failed to enhance the locomotor activity and to induce the characteristic EEG changes. Conclusions: The observed alterations in open field behaviour and quantitative EEG after injection of 2-MeSATP into the NAc may be mostly due to P2 receptor-mediated dopamine release and subsequent receptor activation.

Parathyroid hormone‐related protein plays a major role in the pathogenesis of humoral hypercalcemia of malignancy. Under normal physiological conditions, parathyroid hormone‐related protein is produced in a wide variety of tissues and acts in an autocrine or paracrine fashion. Parathyroid hormone‐related protein and parathyroid hormone bind to and activate the same G‐protein‐coupled receptor. Here we present the structure of the biologically active NH 2 ‐terminal domain of human parathyroid hormone‐related protein(1–34) in near‐physiological solution in the absence of crowding reagents as determined by two‐dimensional proton magnetic resonance spectroscopy. An improved strategy for structure calculation revealed the presence of two helices, His‐5–Leu‐8 and Gln‐16–Leu‐27, connected by a flexible linker. The parathyroid hormone‐related protein(1–34) structure and the structure of human parathyroid hormone(1–37) as well as human parathyroid hormone(1–34) are highly similar, except for the well defined turn, His‐14–Ser‐17, present in parathyroid hormone. Thus, the similarity of the binding affinities of parathyroid hormone and parathyroid hormone‐related protein to their common receptor may be based on their structural similarity.

In the synthesis of cyclic hexapeptides the cyclization step was examined by using different procedures for activation. Three coupling methods were tested for 'head-to-tail'' cyclization of the resin-cleaved peptides in solution: (1) DPPA/NaHCO3; (2) TBTU/HOBt/DIEA and (3) HBTU/HOBt/DIEA. The best results were obtained by using TBTU. Reaction rates were 5 to 70 times faster with the TBTU procedure compared to the DPPA activation and generally complete conversions to cyclopeptides were observed. The kinetics of the cyclizations under high dilution conditions were monitored by HPLC. During the synthesis of the linear peptides the formation of an aminosuccinimide was observed. This by-product was characterized by electrospray MS and NMR.

The short-cut analog of neuropeptide Y (NPY), NPY 1YESK-Ahx-25RHYINKITRQRY-NH2, was synthesized on the amide anchor 5-(4-aminomethyl-3,5-dimethoxyphenoxy)valeric acid (ADPV) attached to polystyrene/1% divinylbenzene by using the 9-fluorenylmethoxycarbonyl/tert-butyl (Fmoc/tBu) strategy. Side-chain-to-side-chain cyclization of residues Glu2 to Lys30 of this linear heptadecapeptide amide was carried out by using different activating reagents under high-dilution conditions of the free peptide. The use of diphenylphosphoryl azide (DPPA) with the addition of triethylamine gave the highest yield of cyclic peptide. Replacing the tertiary amine by dipotassium hydrogen phosphate as an insoluble inorganic base did not improve the cyclization. Complete cyclization was achieved within four hours by using 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) in combination with 1-hydroxybenzotriazol (HOBt) as activating agent, but the separation from excess TBTU resulted in lower yields. Reactions were monitored by HPLC and kinetic investigations were made. The identity and purity of the final product YESK-Ahx-RHYINKITRQRY-NH2 were proven by various analytical methods including atmospheric pressure ionization mass spectrometry (API-MS). The receptor binding constant of the cyclic analog was found to be comparable to that of NPY.