A new GABA-A receptor subtype coupled with Ca++/Cl- synporter modulates aminergic release from rat brain neuron terminals.

The aim of the present study was to give a better characterization of GABA receptors that modulate aminergic release. GABA or muscimol (15 microM) increased basal noradrenaline (3H-NA) release but reduced the following K+-evoked 3H-NA release in the synaptosomes from rat cerebellar cortex. Bicuculline and picrotoxin counteracted these two effects. The same GABA modulation resulted to operate also on dopaminergic and serotoninergic neuron terminals. The increased basal noradrenaline release resulted to be both calcium and chloride dependent and associated with an increased entry of 45Ca++ into the synaptosomes. We therefore advance the hypothesis of an involvement of a Cl-/Ca++ synporter system coupled to the receptor. Baclofen also reduced the K+-evoked 3H-NA release, but did not increase basal 3H-NA release; moreover, the interaction of baclofen G with GABA-B receptors resulted to be associated with the inhibition of 45Ca++ entry into synaptosomes. GABA-B receptors resulted to be present also on serotoninergic but not on dopaminergic neuron terminals. The GABA-C receptor agonist cis-4-aminocrotonic acid (CACA) did not influence either basal or K+-evoked 3H-NA release. These results point to a new type of GABA functional role through a different A-family receptor subtype, coupled with calcium influx in aminergic neuron terminals, modulating aminergic release.

An immunohistochemical analysis of nm23 gene product expression in uveal melanoma.

Uveal melanoma is characterized by an unpredictable clinical course, during which metastatic disease may occur after a prolonged and, at present, undefinable disease-free interval. Because of its relative rarity and the dispersion of cases, the possible genetic alterations implicated in the invasive and metastatic behaviour of this ocular neoplasm have not yet been characterized. The aim of this immunohistochemical retrospective study was to assess the expression of nm23 gene product, proposed to be a metastasis-suppressor gene, in uveal melanoma and to analyse its prognostic significance in relation to the various conventional histopathological parameters, currently considered the major prognostic indicators in this intra-ocular neoplasm. We analysed formalin-fixed paraffin-embedded samples excised from 33 patients with uveal melanoma. Of these, 22 (67%) were positive for monoclonal antibody nm23. This nm23 positivity was inversely associated with scleral invasion level (P = 0.001) and largest tumour diameter (P = 0.02), which represent the two most significant prognostic factors for metastasis. On the other hand, there was no correlation between nm23 expression and other prognostic markers such as cell type, intraocular location or clinical characteristics. These results may suggest a close relationship between nm23 gene expression and metastatic potential of uveal melanomas. In addition, analysis of nm23 gene expression on bioptic tissue may represent an extreme useful prognostic tool for metastatic progression of uveal melanomas.

Influence of prelimbic and sensorimotor cortices on striatal neurons in the rat: electrophysiological evidence for converging inputs and the effects of 6-OHDA-induced degeneration of the substantia nigra.

These studies were designed to investigate whether there are convergent prelimbic and sensorimotor cortical inputs onto striatal neurons in the rat and whether dopaminergic (DA) nigrostriatal fibers regulate these inputs. The influence of the nigrostriatal DA system was assessed in rats with either small or large 6-hydroxydopamine-induced lesions of the substantia nigra. In normal rats 39 out of 74 neurons (52.7%) were excited by stimulation of both the prelimbic and the sensorimotor cortex. No marked change in corticostriatal transmission was evident in rats with small 6-OHDA-induced lesions (defined as 10-35% decrease in [3H]DA uptake in striatal synaptosomes). In rats with large lesions (75-85% decrease in striatal [3H]DA uptake), however, a complete rearrangement of the corticostriatal transmission occurred. This was evident in a decrease of thresholds to obtain cortical responses, by modifications of the discharge properties of striatal neurons receiving input from cortices and by an increase in the number of neurons responding to cortical stimulation. In addition, a significantly higher percentage of striatal neurons responded to stimulation of both prelimbic and sensorimotor cortices in rats with large lesions than in rats with small lesions or in control rats. This data suggests that: (1) no functional separation of prelimbic and sensorimotor cortical inputs occurs in the rat striatum, (2) the nigrostriatal DA system exerts a focusing effect on these inputs, (3) the striatum is actively involved in the integrative processing of descending cortical information.

Age-related changes in rat brain monoamines release: peculiarity of dopamine release.

Our aim has been to investigate the ability of the rat brain to retain its level of neurotransmitter release over life. We have investigated the neurotransmitter release from the rat brain synaptosomes prelabeled with 3H-DA, 3H-NA, or 3H-5HT, and perfused with Krebs-Ringer medium alone (basal release) or containing a high K+, calcium ionophore, tyramine or amphetamine (evoked release). Brain areas have been dissected of animals 45 days and 4, 6, and 11 months old. The results have shown a gradual reduction of the 3H-NA release evoked by a high K+ from 45 days to 6 months, which is stabilized until 11 months of age. The reduction rate has been relatively different from the brain areas investigated (36% for the frontal cortex and 26% for the hippocampus and cerebellar cortex). A similar reduction has been seen with 3H-5HT released from synaptosomes of the frontal cortex, hippocampus, and striatum. Surprisingly, the 3H-DA release that was evoked by high K+ was greater in rats 11 months old than in younger rats; this effect has been seen in synaptosomes from the caudate and the frontal cortex. The calcium ionophore A23187 has shown a releasing picture similar to a high K+. When we analyzed a nonexocitotic, but probably carrier mediated, release (evoked by tyramine or amphetamine), there was reduced release of all of the above neurotransmitters from 45 days to 11 months of age. We presume that there have been adaptive changes in neurotransmitter evoked release due to changes in Ca++ utilization, as inferred from the results from calcium ionophore experiments and carrier performance.(ABSTRACT TRUNCATED AT 250 WORDS)

5HT2-receptors and serotonin release: their role in human platelet aggregation.

Involvement of 5HT2 receptors in human platelet aggregation was assessed by studying the effect of ADP, epinephrine and thrombin on 3H-5HT release from platelets. The release experiments were made with a perfusion method to preserve any compound, released or formed by platelet, from interacting with platelet itself. In these conditions, aggregation does not occur, as confirmed by Scanning Electron Microscopy. These release experiments showed that the platelet activation by such agents is coupled with 5-HT release. The aggregation experiments, made on different aliquots of the same platelet-rich plasma (PRP), showed that the released 5-HT, interacting with its own receptors on platelet activated surface, determines aggregation. In fact, although it is known that 5-HT added to PRP was only able to induce a moderate platelet aggregation, the 5-HT2 antagonist ketanserin counteracted the aggregation induced by ADP, epinephrine and thrombin. These results suggest that a 5HT2 antagonist could be therapeutically important in those pathological states in which serotonin, released by activated platelets, may increase aggregation.

Rat brain alpha 2-pre- and postsynaptic receptors are different or differently modulated?

In order to get better characterization of alpha 2-pre- and postsynaptic noradrenergic receptors in the rat brain, we investigated the alpha 2-receptor changes which take place during a 12-day treatment with the alpha 2-antagonists yohimbine (4 mg/kg) and mianserin (10 mg/kg). These treatments caused a significant increase in the sensitivity of hypothalamic synaptosomes to the inhibitory action of the noradrenergic agonist clonidine on the 3H-noradrenaline release elicited by K depolarization. Frontal cortex alpha 2-autoreceptors were not affected by drug treatments. However, the 3H-p-aminoclonidine (3H-PAC) binding to membranes from hypothalamus or frontal cortex from treated animals was the same as in controls. Changes in neural firing, elicited by the alpha 2-antagonists on noradrenergic neurons, could explain our results. The presynaptic autoreceptors may thus become hypersensitive to counteract the enhanced neurotransmitter release in the hypothalamus, where the noradrenaline is accumulated at the synaptic cleft. In the frontal cortex, where it seems that only 5% of the noradrenergic terminals make synaptic contacts with postsynaptic elements, the alpha 2-autoreceptors are less sensitive to an enhanced neurotransmitter release. Alternatively, they have scarce functional importance because the noradrenaline release is effectively modulated by the inhibitory recurrent locus coeruleus collaterals. At the postsynaptic level, the receptor down-regulation might be prevented by chronic presence of the antagonist drug. Thus the different behavior between pre- and postysynaptic alpha 2-receptors and between alpha 2-receptors of different brain areas may be ascribed to a different modulation rather than to different molecular arrangements.

Long-term treatment with clonidine and alpha-receptors in the brain of normotensive rats.

The aim of the present study was to investigate whether or not changes in rat brain alpha-adrenoceptors take place during chronic treatment with a low dose of clonidine. Male Wistar normotensive rats were treated with clonidine (0.1 mg/kg)i.p. twice daily for 12 days. This treatment caused a significant increase in [3H]clonidine and in [3H]WB4101 binding, respectively, to alpha 2- and to alpha 1-adrenoceptors of the frontal cortex; the levels were 30% for [3H]clonidine and 20% for [3H]WB4101. The Scatchard analysis of data obtained in binding studies indicated that the enhanced binding of two ligands to membranes prepared from chronically clonidine-treated animals, was due to an apparent increase in the number of binding sites. These changes were seen 4 h after administration of the last treatment, before the appearance of the withdrawal syndrome. However, noradrenergic alpha 2-autoreceptors of synaptosomes, from the frontal cortex and hypothalamus of treated animals, were sensitive to the regulatory action of clonidine or of noradrenaline on the [3H]noradrenaline overflow elicited by high K+ as well as on the control animals. On the contrary, the alpha 2-receptors on the serotoninergic nerve terminals from the frontal cortex of treated animals were more sensitive than those of control animals to the action of clonidine or of noradrenaline in counteracting the [3H]5-hydroxytryptamine overflow elicited by high K+. These results suggest that during treatment with clonidine no autoreceptor hyposensitivity to the regulatory action of clonidine or noradrenaline on [3H]noradrenaline overflow elicited by high K+ takes place, but, as a consequence of the diminished noradrenaline availability at the synaptic cleft, the binding of [3H]WBA101 to alpha 1-receptors and of [3H]clonidine to pre- and postsynaptic alpha 2-receptors were significantly elevated in the frontal cortex, a brain areas where the alpha-2-receptors are mainly postsynaptic. Thus, the neurotransmitter concentration in the synaptic cleft may be responsible for the trans-synaptic modulation of the alpha 2-adrenoceptor postsynaptic population. In fact, the alpha 2-adrenoceptors which are presynaptically located on the serotoninergic terminals, but are postsynaptic in relation to the noradrenergic neurons, also show increased sensitivity after chronic clonidine treatment.

Etomidate inhibits prolactin release but not through a dopaminergic mechanism.

A short-acting non-barbiturate intravenous general anaesthetic, etomidate, when administered i.v. lowered serum prolactin levels in the rat. The effect was evident at rest, after surgical stress or after injection of 5-hydroxytryptophan. Haloperidol-induced hyperprolactinaemia was not modified. Administered via the intracerebroventricular route, etomidate strongly reduced serum prolactin levels in unanesthetized rats. Studies performed on superfused synaptosomes from brain areas rich in dopaminergic nerve endings did not show any influence of the etomidate, 10(-7) M-10(-6) M, on [3H]dopamine release. A peculiar GABA-like mechanism and/or possible interplay with serotonergic control of the prolactin release may be postulated in order to explain the suppressive effects of the drug on secretion of lactotrophs.

Effects of naloxone on the secretion of prolactin and corticosterone induced by 5-hydroxytryptophan and a serotonergic agonist, mCPP.

The effects of naloxone, an opiate "pure" receptor antagonist, on the release of prolactin and corticosterone in the rat were studied following the administration of the serotonin precursor 5-hydroxytryptophan or the serotonin receptor agonist (-) -m-chlorophenylpiperazine. Naloxone clearly antagonizes the release of prolactin induced by 5-hydroxytryptophan administered alone at a dosage of 50 mg/Kg/b.wt. or at dosage of 30 mg/Kg/b.wt. preceded 60 minutes before injection by the administration of the serotonin uptake blocker fluoxetine. The opiate antagonist does not modify the increase in blood level of prolactin induced by (-) -m-chlorophenylpiperazine. Naloxone itself does not reduce the increase in plasma level of corticosterone induced by 5-hydroxytryptophan, 5-hydroxytryptophan+fluoxetine or (-)-m-chlorophenylpiperazine. The results suggest that endogenous opioids may be involved in the increase in serum level of prolactin induced by 5-hydroxytryptophan and also indicate the existence of different serotonergic neurotransmitter circuits capable of modulating the release of prolactin and corticosterone. A mutual interplay between serotonergic and opiate neurons may be involved in controlling the release of prolactin, but such an interplay does not seem to occur in the secretion of corticotrophin-releasing hormone.

Dopamine biosynthesis is regulated by the amine newly recaptured by dopaminergic nerve endings.

The synthesis of dopamine from labeled tyrosine (but not from labeled DOPA) in rat striatal synaptosomes was effectively inhibited by exogenous dopamine only when the amine was allowed to enter the nerve endings. In the presence of the uptake blocker nomifensine, extracellular dopamine was almost inactive. The evolution of 14CO2 from [14C]tyrosine was consistently higher when synaptosomes were 'incubated' in the presence of nomifensine than in its absence. This effect disappeared when synaptosomes were 'superfused' with labeled tyrosine (with or without nomifensine) in conditions in which dopamine reuptake cannot occur. The monoaminoxidase inhibitor pargyline inhibited 14CO2 evolution from [14C]tyrosine. However, the effect was almost abolished if dopamine reuptake was prevented (by nomifensine or in superfusion). Our results suggest that dopaminergic nerve endings do not possess autoreceptors controlling dopamine synthesis. In the present paper it is proposed that the regulation of dopamine synthesis occurs through inhibition of tyrosine hydroxylase, according to the classical and end-product concept; however, the function of 'end-product' would be primarily exerted by the amine newly taken up by the nerve terminals.

Evidence for a similar compartmentation of recaptured and endogenously synthesized dopamine in striatal synaptosomes.

The aim of the present study was to compare the release pattern of [3H]dopamine ([(3H]DA) originated from [3H]tyrosine or by uptake in striatal synaptosomes. Synaptosomes prelabeled either with [3H]DA or with [3H]tyrosine were superfused in three conditions stimulating DA release by different mechanisms: (1) depolarization with high K+ (2) inversion of the NA+ gradient across the plasma membrane; (3) exposure to d-amphetamine. Since DA contained in different pools may exit from nerve endings by different processes, DA release was analyzed in the presence or in the absence of nomifensine which allows discrimination between carrier-mediated and carrier-independent processes. The pattern of DA release in the three conditions tested was idential, whether [(3)H]DA originated from synthesis or from uptake. Nomifensine did not affect the high-K+-induced release and inhibited that induced by the other two stimuli. The results suggest that newly synthesized and recaptured DA have similar compartmentation in nerve endings.