The gamma(2)-MSH peptide mediates a central analgesic effect via a GABA-ergic mechanism that is independent from activation of melanocortin receptors.

Using the latency for tail-flick after thermal stimulation we have assessed the effects of alpha-, gamma(1)- and gamma(2)-MSH on nociceptive threshold in the mice. Intracisternal injections of gamma(2)-MSH induced a distinct analgesia, while gamma(1)-MSH in the same doses gave only a minor analgesia. Intracisternal alpha-MSH instead gave a short-term hyperalgesia. The effect of gamma(2)-MSH was not blocked by any of the MC(4)/MC(3)receptor antagonist HS014, naloxone or by the prior intracisternal administrations of gamma(1)-MSH. However, the gamma(2)-MSH analgesic response was completely attenuated by treating animals with the GABA(A)antagonist bicuculline. The gamma(2)-MSH analgesic effect was moreover additive to the analgesia afforded by muscimol and ethanol, but not to that afforded by diazepam. In addition both gamma(1)- and gamma(2)-MSH induced moderate catalepsy, but could at the same time attenuate haloperidol induced catalepsia. We conclude that gamma(2)-MSH mediates a central analgesic effect via GABA-receptor dependent pathway that is distinct from melanocortic- and opioid-receptors. Moreover, the mechanism for gamma(2)-MSH's analgesic effect appears to be distinct from that causing moderate catalepsia by gamma-MSH's.

Behavioural responses of gamma-MSH peptides administered into the rat ventral tegmental area.

The behavioural effects induced by alpha-, gamma1- and gamma2-MSH peptides (0.3 and 3 nmole per rat) injected into the left ventral tegmental area (VTA) of rats were compared. alpha- and gamma1-MSH caused grooming of comparable magnitude, and also additional vertical activity (rearing). By contrast gamma2-MSH caused a moderate but stable catalepsy, and practically no grooming. Moreover, intra-VTA pre-treatment with gamma2-MSH, 15 min prior to intra-VTA gamma1-MSH, markedly attenuated both the gamma1-induced grooming and vertical activities. The differences in the behavioural response of the MSH peptides indicate that they act differentially on MC receptors in the VTA.

Evaluation of behavioural effects of neural melanocortin receptor antagonists injected ICV and in VTA in rats.

The natural melanocortic peptides are known to exert a variety of effects after central administration. Recently, we discovered the first potent and selective substances for the MC4 receptor, i.e. HS964 and HS014. We found HS964 to be an antagonist for the MC1, MC3, MC4 and MC5 receptors in vitro. HS014 is an antagonist for the MC3 and MC4 receptors and a partial antagonist for the MC1 and MC5 receptors. We injected alpha-MSH and these substances, both intracerebroventricular (ICV) and in the ventral tegmental area (VTA) in rats and scored several behavioural effects. The results show that alpha-MSH caused intensive grooming which was antagonized by pre-treatment of both HS014 and HS964. The data give further support to the hypothesis that it is the MC4 receptor which mediates grooming in rodents. The grooming effects of alpha-MSH were more pronounced after intra-VTA administration compared to the ICV administration. Both alpha-MSH, HS014 and HS964 caused an increase in vertical activity of the rats after intra-VTA administration but not after ICV administration. Horizontal activity was virtually not affected by the administration of the peptides. The data indicate that the neural MC3 and MC4 receptors are not likely to be an important mediators of locomotor activity in rats.

Modification of swelling-contraction-aggregation processes in rat muscle mitochondria by the 1,4-dihydropyridines, cerebrocrast and glutapyrone, themselves and in the presence of azidothymidine.

The influence of the 1,4-dihydropyridines (DHPs), water-soluble glutapyrone available as sodium, potassium and ammonium salts of 2-(2,6-dimethyl-3,5-diethoxycarbonyl-1,4-DHP-4-carboxamide)glutaric acid, from one side, and a lipophylic cerebrocrast, 2-propoxyethyl 2,6-dimethyl-4-(2-difluoromethoxyphenyl)-1,4-DHP-3,5-dicarboxylate, from the other side, on partially damaged mitochondria of the Wistar rat hindlimb muscle was also studied. The following tests were made: (1) rates of endogenous respiration and substrate (succinate) oxidation and oxidative phosphorylation; (2) rates and amplitudes of high-amplitude swelling and contraction after the addition of ATP, ADP and succinate to the previously swollen mitochondria and (3) rate of reversible self-aggregation of mitochondria isolated in salt media after ATP-induced contraction without and in the presence of azidothymidine (AZT). Cerebrocrast (10-100 microM) partially normalized the endogenous respiration rate and slightly augmented the respiration rate after the addition of succinate and to lesser extent ADP. Cerebrocrast in a concentration-dependent manner (2.5-50 microM) increased (two-fold at 20-50 microM) the active contraction amplitude of swollen mitochondria, induced by single or repeated additions of ATP. The influence of cerebrocrast on the ADP- and succinate-induced contractions was less obvious. Unlike cerebrocrast glutapyrone caused a reduction of the ATP-induced contraction amplitude (two-fold at 0.5-5.0 mM), not impairing the mitochondrial contraction ability in response to ATP or succinate. Pre-exposure to 2.5 mM glutapyrone resulted in at least a 10-fold inhibition of the reversible aggregation rate in the presence of 99 and 198 microM AZT. The results suggest the usefulness of further study of cerebrocrast and glutapyrone in preventing AZT-induced and some other mitochondrial myopathies.

Thymopentin antagonizes stress-induced changes of GABA/benzodiazepine receptor complex.

Intraperitoneal administration of thymopentin, a thymopentin II-derived pentapeptide, had no stable and evident effect in the two anxiety models (elevated plus-maze and licking-conflict test) studied. However, in the elevated plus-maze test thymopentin antagonized the behavioral effects of DMCM, a beta-carboline derivative with anxiogenic properties. Further, it was demonstrated that the licking-conflict test procedure itself produced a significant elevation of plasma corticosterone levels, increased the number of [3H]flunitrazepam and decreased the number of [3H]muscimol binding sites in rat hippocampus. The forced-swimming stress similarly to the licking-conflict test also caused an increase in hippocampal [3H]flunitrazepam binding sites. Although ineffective behaviorally in the tests for anxiety, thymopentin pretreatment effectively reversed the changes in corticosterone levels caused by the licking-conflict test. Moreover, it normalized the changed number of benzodiazepine and GABA receptors after stressful stimuli. It is well known that not all anxiolytic drugs (i.e. buspirone) are equally active in behavioral tests for anxiety. According to our data we propose that thymopentin has stress-protective activity. As in vivo and in vitro thymopentin did not change [3H]-flunitrazepam and [3H]muscimol binding, the direct effect of this peptide on the GABA-benzodiazepine-Cl- ionophore receptor complex is unlikely. The action of this peptide on GABA release and/or metabolism can be suggested.

Smooth-muscle effects of C-terminal tripeptide of substance P.

The effects of the C-terminal tripeptide fragment of substance P(SP-C-TP) and its interactions with substance P(SP) were studied on isolated smooth-muscle preparations (rat musculus anococcygeus and vas deferens and rabbit ear central artery). Like SP, SP-C-TP (1 x 10(-8) M-1 x 10(-4)M) potentiated contractions of vas deferens and musculus anococcygeus elicited by low frequency electrical stimulation (LFES), the effect of the fragment being much weaker than that of the neuropeptide. The potentiating effect of SP was strongly reduced by SP-C-TP. The fragment slightly decreased the noradrenaline (NA)-induced contractions of vas deferens only. When the pharmacological agents were applied extralumenally only high SP concentrations (1 x 10(-7)M and 1 x 10(-6)M) significantly potentiated the LFES-evoked smooth-muscle contractions. SP-induced potentiation was abolished by SP-C-TP. Intralumenal administration of the neuropeptide and its fragment always led to a marked inhibition of the LFES contractile effects. The observed effects suggest that SP-C-TP acts as a competitive dualist of SP. The data is interpreted in terms of the hypothesis that the neuropeptide fragments might perform as a natural antagonist of the peptides and may control their effects.

Comparative studies on the central effects of the angiotensin II analogue (Sar1 azaVal3 Ile8) AT II.

The effects of the newly-synthesized AT II analogue (Sar1 azaVal3 Ile8) AT II were investigated in comparison with the octapeptide AT II and the analogue saralazin (Sar1 Ala8) AT II, using intracerebroventricular administration, with respect to the following parameters: the level of biogenic monoamines (DA, NA and 5-HT) and the metabolites HVA and 5-HIAA in mouse forebrain; the behaviour of the animals--cataleptogenic actions of mice, PTZ convulsive--seizure threshold in mice, apomorphine stereotypy in rats and behaviour of rats in a conflict situation. The analogue (Sar1 azaVal3 Ile8) AT II, unlike saralazin and AT II, was found to induce a rise in the NA and 5-HT levels, causing also catalepsy that is different from the catalepsy induced by saralazine, AT II and haloperidol, because of its rapid onset and decline; it increases the PTZ convulsive--seizure threshold and reduces the number of punished responses to the conflict drinking test (anxiomimetic effect) in a dose 20 times lower than the dose inducing the remaining effects. This effect was antagonized by saralazine. It is concluded that the newly-synthesized analogue (Sar1 azaVal3 Ile8) AT II induces effects similar to those caused by AT II, being at the same time different to a certain extent from the effects (quantitative and qualitative) of octapeptide AT II.

Comparative studies on the central effects of angiotensin II and of its fragments.

Comparative studies were carried out of the central effects of the octapeptide angiotensin II (AT II) and of its fragments: C-terminal hexapeptide (AT 3-8), middle tetrapeptide (AT 3-6) and initial tripeptide (AT 1-3). The experiments were carried out with respect to the cerebral level of the biogenic amines DA, NA, 5-HT and their metabolites HVA and 5-HIAA in intact mice and in mice pretreated with haloperidol, as well as with respect to the animals' behaviour (haloperidol catalepsy, apomorphine stereotypy, unconditioned jumping reaction, hexobarbital sleep and the threshold of convulsive seizures induced by times intravenous infusion of pentylenetetrazol). The fragments studied were found to manifest activity in the tests used. In some respects this activity is very similar to the activity of AT II. There are also effects which differ from those of AT II, as well as effects which are entirely opposite in some respects, which makes it possible to examine some of these substance as its potential natural antagonists. All this shows that the biological activity of AT II in the brain undergoes a number of qualitative and quantitative changes when its molecule broken to produce peptides with shorter chains. In the effects observed AT II and its fragments interact predominantly with the DA-ergic transmission in the brain.

Central effects of angiotensin II, its fragment and analogues.

The effects of the octapeptide angiotensin II (AT II), its fragment Ile8 AT3-8 and the analogues Sar1 Ala8 AT II, Ala8 AT II and Ile8 AT II were studied with respect to: the level of biogenic amines (DA, 5-HT and their metabolites HVA and 5-HIAA) in the forebrain; the behaviour of the animals--haloperidol catalepsy, apomorphine stereotypy, unconditioned jumping reaction (UJR), convulsive threshold. Good correlation was found between the biochemical and behavioural effects. The fragment of AT II where phenylalanine is substituted at the C-terminal by Ile reduces the haloperidol-increased content of HVA, potentiates apomorphine stereotypy and reduces catalepsy, whereas the AT II analogues (where the C-terminal phenylalanine is substituted by Ala, and the N-terminal--by Sar) potentiate the effect of haloperidol increasing the HVA content, reduce apomorphine stereotypy and potentiate catalepsy; saralasine independently applied induces brief catalepsy; AT II, its fragment and analogues inhibit UJR, in combination with amphetamine and PTZ this effect becomes deeper; the duration of hexobarbital sleep is increased. The peptides investigated increase the convulsive threshold. The results show that the hexapeptide fragment has preserved the effects of AT II, whereas in the analogues (with changed C- and N-terminals) they are changed. The results obtained may be explained with the modulating influence of AT II-receptors on the DA-ergic receptors in the brain structures with which AT II and its fragment and analogues enter in contact.