Pharmacological profile of various kappa-agonists at kappa-, mu- and delta-opioid receptors mediating presynaptic inhibition of neurotransmitter release in the rat brain.

1. The potency, relative efficacy and selectivity of a series of kappa-opioid receptor agonists at the mu-, delta- and kappa-opioid receptors mediating inhibition of electrically-induced (radiolabelled) neurotransmitter release from superfused rat brain slices was determined. 2. With regard to their potencies at kappa-receptors mediating inhibition of striatal [3H]-dopamine release, the highest pD2 value (8.7) was found for bremazocine and the lowest (7.1) for U50488; the pD2 values for ethylketocyclazocine (EKC), tifluadom, U69593 and PD117302 were between 8.0 and 8.3. There were no marked differences between the relative efficacies of the kappa-agonists (maximum inhibition being 60-70%). In contrast to the other kappa-agonists, at a concentration of 1 microM, PD117302 caused a significant (25-40%) increase of the spontaneous efflux of tritium. 3. None of the kappa-agonists significantly affected striatal [14C]-acetylcholine (ACh) release, with the exception of a slight inhibitory effect of EKC. The delta-receptor-mediated inhibitory effect of [D-Ala2, D-Leu5]enkephalin (DADLE) on [14C]-ACh release was antagonized in a concentration-dependent manner by bremazocine (0.1 and 1.0 microM) and also partially by EKC (1 microM), but not by the other kappa-agonists. The pA2 value for bremazocine as an antagonist at the delta-receptors involved was 8.0, compared to 7.6 for naloxone. 4. None of the kappa-agonists significantly affected cortical [3H]-noradrenaline (NA) release, with the notable exception of tifluadom, which strongly inhibited release by activating mu-receptors. The mu-receptor-mediated inhibitory effect of Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol (DAMGO) on [3H]-NA release was antagonized in a concentration-dependent manner by bremazocine and EKC, but not by the other K-agonists. The pA2 value for bremazocine as an antagonist at the mu-receptors involved was 8.2, compared to 8.6 for naloxone. 5. Thus, whereas U69593 and PD1 17302 display high potency and selectivity towards K-opioid receptors, the potent benzomorphan K-agonists bremazocine and EKC also appear to be strong mu-opioid receptor antagonists.

Selectivity of various opioid peptides towards delta-, kappa; and mu-opioid receptors mediating presynaptic inhibition of neurotransmitter release in the brain.

The selectivity of a series of opioid peptides towards the mu-, delta- and kappa-opioid receptors mediating differential inhibition of electrically-induced neurotransmitter release from rat brain slices was studied, viz. cortical [3H]noradrenaline release (inhibited via mu-receptors), striatal [3H]dopamine release (inhibited via kappa-receptors) and striatal [14C] acetylcholine release (inhibited via delta-receptors). The highest affinity pD2 7.4) and selectivity towards mu-receptors was exhibited by Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol (DAGO), whereas [D-Pen2, D-Pen5]enkephalin (DPDPE) was found to be the most selective delta-receptor agonist (pD2 7.3). Also the hexapeptides [D-Ser2]Leu-enkephalin-Thr (DSLET) and [D-Thr2]Leu-enkephalin-Thr (DTLET) showed a relatively high selectivity and, in addition, a high affinity (pD2 8.2-8.4) for delta-opioid receptors. Both dynorphin(1-13) and dynorphin(1-8) exhibited a high affinity for kappa-receptors (pD2 resp. 8.3 and 8.0), but the latter was far less selective. Both of the dynorphin A-related peptides showed affinity to mu-receptors (pD2 6.7-6.8), but dynorphin(1-8), in contrast to dynorphin(1-13), also displayed a high affinity to delta-receptors (pD2 7.6).

Effects of neostigmine, edrophonium, 4-aminopyridine and their combinations.

A study was carried out in the in-vitro rat phrenic nerve hemidiaphragm preparation to find a fast and strong working antagonist of non-depolarizing muscle relaxants. We studied neostigmine, edrophonium, 4-aminopyridine and their combinations from the cumulative concentration response curves of pancuronium. Neostigmine, edrophonium and 4-aminopyridine all antagonized the effect of pancuronium. Neostigmine and 4-aminopyridine did potentiate each other as did neostigmine and edrophonium. The combination of edrophonium and 4-aminopyridine showed less total effect than the addition of their individual effect. We conclude that the mechanisms of action of neostigmine and edrophonium are different.

Functional activity of raphe neurons transplanted to the hippocampus and caudate-putamen. An immunohistochemical and neurochemical analysis in adult and aged rats.

Adult (3-month-old) and aged (28-month-old) rats that had been pretreated with 5,7-DHT in both lateral ventricles received grafts of cell suspensions taken from the RR or MR regions taken from the embryonic stages E12-21. These cell suspensions were implanted unilaterally into the rostral part of the hippocampus or the caudate-putamen for immunohistochemical and neurochemical studies. MR and RR cell suspensions have the potency to regenerate serotonergic fibers in the previously denervated adult and aged hippocampus and caudate-putamen. The RR cell suspension, however, also showed outgrowth of other transmitter-specific neuronal systems, specifically noradrenaline and substance P. To evaluate the functional activity of the serotonergic reinnervation, we have combined immunohistochemistry and neurotransmitter release studies on adjacent hippocampus slices of adult rats. Results showed that after a survival time of 10 weeks, the serotonergic innervation of the hippocampus was greatly restored and, moreover, that the K+-induced Ca2+-dependent release of 5-HT amounted to about 80% of normal values. There appeared to be a striking similarity between the immunohistochemical and neurochemical data regarding the increase in the number of newly formed serotonergic fibers, the increase of the release of radiolabeled 5-HT, and the extent of the outgrowth in the hippocampus.

Kappa- and delta-opioid receptor agonists differentially inhibit striatal dopamine and acetylcholine release.

At least three different families of endogenous opioid peptides, the enkephalins, endorphins and dynorphins, are present in the mammalian central nervous system (CNS). Immunocytochemical studies have demonstrated their localization in neurones, which supports the view that these peptides may have a role as neurotransmitter or neuromodulators. However, the target cells and cellular processes acted upon by the opioid peptides are still largely unknown. One possible function of neuropeptides, including the opioid peptides, may be presynaptic modulation of neurotransmission in certain neuronal pathways, for example, by inhibition or promotion of neurotransmitter release from the nerve terminals. Here we report that dynorphin and some benzomorphans potently and selectively inhibit the release of (radiolabelled) dopamine from slices of rat corpus striatum, by activating kappa-opioid receptors. In contrast, [Leu5]enkephalin and [D-Ala2, D-Leu5]enkephalin selectively inhibit acetylcholine release by activating delta-opioid receptors.

Pharmacological characterization of presynaptic alpha-adrenoceptors modulating [3H]noradrenaline and [3H]5-hydroxytryptamine release from slices of the hippocampus of the rat.

The experiments served to characterize the receptors mediating the inhibitory effect of alpha-adrenergic drugs on K+ (20 mM)-induced [3H]noradrenaline (NA) and [3H]5-hydroxytryptamine ([3H]5-HT) release from slices of the dorsal part of rat hippocampus. Dose-response curves were constructed using the cumulative dose-response technique (Frankhuyzen and Mulder, 1981). All of the adrenergic agonist drugs examined inhibited the K+-induced [3H]NA release. NA appeared to have the highest intrinsic activity followed by adrenaline. Clonidine and adrenaline had similar intrinsic activities, while that of oxymetazoline was lowest. The highest pD2 values were observed for oxymetazoline and clonidine, being slightly higher than that of adrenaline followed by NA. By far the lowest pD2 values was observed for phenylephrine. With the exception of phenylephrine, all of the agonists also inhibited the K+-induced [3H]5-HT release. NA, adrenaline and oxymetazoline appeared to have similar intrinsic activities, while that of clonidine was considerably lower. The pD2 values of NA and adrenaline were not significantly different but were somewhat lower than those of oxymetazoline and clonidine. Similar antagonistic effects of phentolamine and yohimbine were observed with respect to the adrenergic inhibition of K+-induced [3H]NA and [3H]5-HT release. Prazosin, however, appeared to be ineffective in both instances. It is concluded from these results that the presynaptic adrenergic inhibiton of [3H]NA as well as [3H]5-HT release is mediated by alpha 2-adrenoceptors located on noradrenergic and serotonergic varicosities, respectively. Furthermore, our data suggest that these alpha 2-adrenoceptors are not pharmacologically identical.

Pharmacological characterization of presynaptic alpha-adrenoceptors in the nucleus tractus solitarii and the cerebral cortex of the rat.

The presynaptic alpha-adrenoceptors mediating inhibition of K+-induced release of [3H]noradrenaline (NA) from superfused slices and synaptosomes obtained from the nucleus tractus solitarii (NTS) region were characterized pharmacologically and compared to those in the cerebral cortex. Clonidine concentration-dependently (pD2 7.80) reduced the K+-induced [3H]NA release from slices and synaptosomes from the nucleus tractus solitarii of both normotensive and spontaneously hypertensive rats; 10(-6) M clonidine inhibited the [3H]NA release induced by 10 mM K+ to 40% of control. This inhibitory effect of clonidine was antagonized by the alpha2-adrenoceptor antagonist yohimbine (10(-7) M), but not by the alpha1-adrenoceptor antagonist prazosin (10(-7) M). The rank order of apparent affinities of agonists for the presynaptic alpha-adrenoceptors mediating inhibition of [3H]NA release from slices of nucleus tractus solitarii induced by 20 mM K+, was: oxymetazoline greater than clonidine greater than adrenaline greater than alpha-methylnoradrenaline greater than noradrenaline much greater than methoxamine. A similar rank order was found for cerebral cortex slices. The rank order of intrinsic activities in both brain regions was found to be: noradrenaline approximately equal to alpha-methylnoradrenaline approximately equal to adrenaline greater than clonidine greater than oxymetazoline much greater than methoxamine. The data indicate that presynaptic alpha-adrenoceptors mediating inhibition of NA release both in the nucleus tractus solitarii and the cerebral cortex belong to the alpha2-type. The presynaptic alpha2-adrenoceptors in the two brain regions appear to be similar both functionally and pharmacologically. It is suggested that these receptors in the nucleus tractus solitarii may play a role in the hypotensive effect of clonidine.

A cumulative dose-response technique for the characterization of presynaptic receptors modulating [3H]noradrenaline release from rat brain slices.

A cumulative dose-response technique was developed for the characterization of presynaptic receptors involved in the modulation of [3H]noradrenaline (NA) release from rat hippocampus slices, using continuous K+ (20 mM) depolarization. The results obtained with this technique were compared with those obtained using a repetitive K+ stimulation procedure. The release of [3H]NA induced by continuous K+ stimulation as well as that caused by repetitive K+ stimulation was strongly Ca2+-dependent and consisted for more than 90% of unmetabolized [3H]NA. Using continuous K+ stimulation it was demonstrated that the presynaptic inhibition of 3H-NA release by exogenous NA reached a maximum 10 min after addition of NA. The inhibitory effect of NA appeared to be independent of the time of addition, suggesting that the sensitivity of the presynaptic alpha-adrenoceptors remained unchanged during the experiment. Cumulative dose-response curves were recorded by the successive addition, at 10 min intervals, of increasing concentrations of NA. It was shown that continuous stimulation and repetitive K+ stimulation were basically similar with regard to the characteristics of the resulting [3H]NA release as well as its presynaptic alpha-adrenoceptor-mediated modulation by exogenous NA. However, the cumulative dose-response technique, which can be carried out only using continuous K+ stimulation, makes it possible to determine more rapidly and also more accurately the apparent affinities and intrinsic activities of drugs towards receptors involved in the modulation of neurotransmitter release from brain slices.

Noradrenaline inhibits depolarization-induced 3H-serotonin release from slices of rat hippocampus.

The depolarization (26 mM K+)-induced release of 3H-serotonin and 3H-noradrenaline from slices of rat hippocampus was studied with a superfusion method. Exogenous NA (in the presence of 10 muM desipramine) inhibited 3H-5-HT release (EC50 3 X 10(-7) M) as well as 3H-NA release (EC50 10(-7) M) by more than 70%. Both of these effects were competitively antagonized by phentolamine, but not by propranolol. It is tentatively suggested that the inhibitory effect of NA on 3H-5-HT release from hippocampus slices reflects the activation of postsynaptic alpha-receptors which are localized on serotonergic nerve terminals.