Bromocriptine inhibits incorporation of [3H]thymidine into rat pituitary tumor cells

The effects of bromocriptine on GH3 pituitary tumor cell [3H]thymidine incorporation were studied. Cells were grown in the presence of bromocriptine, then exposed to a short-term pulse of [3H]thymidine in serum-free medium containing deoxycytidine (10 microM) to prevent deoxythymidine triphosphate (dTTP) pooling. After 48 h exposure to bromocriptine, basal prolactin (PRL)-secretion during 45 min was inhibited by 50% by 10 microM bromocriptine and thyroid releasing hormone-induced PRL stimulation was suppressed. Incorporation of radiolabelled thymidine into acid-precipitable DNA increased progressively from 15 to 60 min and was abolished by simultaneous incubation with excess unlabelled thymidine (100 microM). Bromocriptine (10 microM) inhibited incorporation of 5-50 microM [3H]thymidine, but this was not reversed by simultaneous incubation with metoclopramide (10 microM). Aminopterin, an inhibitor of endogenous de novo DNA synthesis, stimulated [3H]thymidine incorporation twofold and this increased DNA salvage pathway activity was also blocked by bromocriptine. As incorporation of [3H]thymidine into acid-soluble cell nucleotides was also inhibited by bromocriptine, the data suggest that in these cells the drug inhibits thymidine kinase activity, a salvage pathway of DNA synthesis.     

Modulation of thymidine incorporation by κ-opioid ligands in rat spinal cord-dorsal root ganglion co-cultures

β-Endorphin, met-enkephalin and several μ-selective opioid agonists were shown to decrease thymidine incorporation into DNA in various neural cell cultures. We now report that the κ-selective opioid agonists U50488, U69593 and MR2034 modulate [³H]thymidine incorporation into DNA in rat spinal cord-dorsal root ganglion co-cultures. U50488 at 10 μM increased by 60% thymidine incorporation in 6-day-old cultures. The thymidine incorporation induced by U50488 was blocked by the κ-selective antagonist nor-binaltorphimine, as well as by pertussis toxin and LiCl U50488 treatment stimulated phosphatidylinositol turnover by three-fold compared with untreated controls. These findings suggest that κ-opioid agonists modulate DNA synthesis in spinal cord-dorsal root ganglion co-cultures through a mechanism which involves pertussis toxin-sensitive GTP-binding proteins, as well as activation of phosphatidylinositol turnover.     

Heparin inhibits the growth of astrocytes in vitro

Heparin at a concentration of 100 μg/ml, inhibits neonatal rat astrocyte proliferation by 45%, and [³H]thymidine incorporation into DNA by 55% when they are stimulated with 2% fetal bovine serum (FBS) in culture. Higher serum concentrations up to 10% FBS decreased this inhibition to 20% and 29% respectively. Inhibition of [³H]thymidine incorporation by heparin is dose-dependent with maximal inhibition at 100 μg/ml, but 39% inhibition is still seen at 1 μg/ml on stimulation by 5% FBS. Heparin or heparin-like molecules in the extracellular matrix of brain capillary walls may be important in the regulation of astrocyte growth in vivo.     

The calcium-dependent [H]acetylcholine release from synaptosomes of brown trout (Salmo trutta) optic tectum is inhibited by adenosine A1 receptors: Effects of enucleation on A1 receptor density and cholinergic markers

Presynaptic inhibition is one of the major control mechanisms in the CNS. Previously we reported that A₁ adenosine receptors are highly concentrated in the brain, including optic tectum, of trout and that they inhibited the release of glutamate. The optic tectum is heavily innervated by cholinergic nerve terminals. We have investigated whether A₁ receptors inhibit the presynaptic release of acetylcholine and whether the inhibition is triggered by calcium. The release of [³H]ACh evoked by 30 mM KCl was Ca²⁺ dependent and it was dose-dependently inhibited by the A₁ adenosine receptor agonist 2-chloro-N⁶-cyclopentyladenosine (CCPA) ranging between 10 nM to 100 μM. The maximum of inhibition was reached at 10 μM. The A₁ receptor antagonist 8-cyclopentyltheopylline (CPT, 10 μM), reversed almost completely the inhibition induced by CCPA 10 μM. In Fura-2/AM loaded synaptosomes, K⁺ depolarization raised [Ca²⁺]_i by about 64%. CCPA (10 μM) reduced the K⁺-evoked Ca²⁺ influx increase by about 48% and this effect was completely antagonised by CPT 10 μM. Synaptosome pretreatment with different Ca²⁺ channel blockers differently affected K⁺-evoked Ca²⁺ influx. This was not significantly modified by nifedipine (1 μM, L-type blocker) nor by ω-agatoxin IVA (0.3 μM, P/Q-type blocker), whereas about 50% reduction was shown by 0.5 μM ω-conotoxin GVIA (N-type blocker). Neurochemical parameters associated with cholinergic transmission and the density of A₁ adenosine receptors were measured in the trout optic tectum 12 days after unilateral eye ablation. A significant drop of both acetylcholinesterase (AChE) activity (24%) and choline acetyltransferase (CAT) activity (32%) was observed in deafferentated optic tectum, whereas the high affinity choline uptake did not parallel the decrease in enzyme activity. Eye ablation caused a marked decrease (43%) of A₁ receptor density without changing the affinity. The K⁺-evoked release of [³H]ACh from synaptosomes of deafferentated was not modify as well as the efficacy of 10 μM CCPA in decreasing [³H]ACh release was not apparently modified.     

On the role of calcium ions in the presynaptic alpha-receptor mediated inhibition of [H]noradrenaline release from rat brain cortex synaptosomes

Rat brain cortex synaptosomes, previously labeled by incubation with [³H]noradrenaline ([³H]NA) were continuously superfused with Krebs-Ringer media. Release of [³H]NA was induced by superfusion with medium containing either 15 mM K⁺, 20 μM veratrine or 1 μM of the calcium-ionophore A 23187 and was strongly dependent on the concentration of Ca²⁺ in the medium. Noradrenaline (1μM, in the presence of the uptake inhibitor desipramine) inhibited K⁺-induced [³H]NA release by activation of presynaptic alpha-receptors. When the Ca²⁺-concentration in the medium was reduced, or the Mg²⁺-concentration increased, [³H]NA release appeared to be more susceptible to alpha-receptor mediated inhibition.Noradrenaline (1 μM) inhibited [³H]NA release induced by 15 mM K⁺, in the presence of 0.075 Ca²⁺ and 10 mM Mg²⁺, by 86%. Veratrine-induced release was also inhibited by alpha-receptor activation. However, [³H]NA release induced by the calcium-ionophore was not affected by alpha-receptor agonists. These results strongly support the view that alpha-receptor activation results in a decrease of the availability of Ca²⁺ for stimulus-secretion coupling processes. Presumably this is effected by an inhibition of voltage-sensitive calcium channels in the neuronal membrane associated with neurotransmitter release.     

Presynaptic effect of 7-OH-DPAT on evoked [H]-acetylcholine release in rat striatal synaptosomes

The objective of the present experiments was to study the presynaptic effect of 7-hydroxy-N,N-di-n-propyl-2-aminotetraline (7-OH-DPAT, a D₂-like dopamine receptor agonist) on [³H]-acetylcholine ([³H]-ACh) release induced by potassium (15 mM, 25 mM and 60 mM), potassium channel-blockers (4-aminopyridine, 4-AP; tetraethylammonium, TEA and quinine) and veratridine to gain insight into the mechanisms involved in the activation of the D₂ dopamine-receptor subtype located at striatal cholinergic nerve terminals. 7-OH-DPAT (1 μM) inhibited the evoked [³H]-ACh release induced by K⁺ 15 mM in a similar percentage than that obtained during basal conditions (30% and 27%, respectively). Nevertheless, in the presence of 25 mM and 60 mM of K⁺ the inhibitory effect of 7-OH-DPAT was completely abolished. 4-AP (1–100 μM) and TEA (1 and 5 mM) significantly enhanced [³H]-ACh release, showing 69.32%±7.60% (P<0.001) and 52.27%±5.64% (P<0.001), respectively, at the highest concentrations tested. In these conditions, 7-OH-DPAT (1 μM) inhibited the release induced by potassium channel-blockers 25–27%. Quinine (0.1–1 μM) did not alter [³H]-ACh release either in the presence or absence of 7-OH-DPAT. Veratridine 10 μM evoked [³H]-ACh release in the presence of a low-calcium medium, but in such conditions 7-OH-DPAT (1 μM) did not modify the neurotransmitter release in the absence or presence of veratridine. Present data indicate that activation of the presynaptic D₂ dopamine receptor inhibits the [³H]-ACh release by increasing K⁺ conductance, as high K⁺ concentrations abolished the inhibitory control of 7-OH-DPAT on [³H]-ACh release. This effect could be mediated by potassium channels different from those sensitive to 4-AP, TEA and quinine. In addition, the presynaptic D₂ dopamine-receptor activation seems to not involve changes in intracellular Ca²⁺.     

Dopaminergic regulation of prolactin and growth hormone synthesis in rat pituitary tumor cells

The dopaminergic regulation of prolactin (PRL) and growth hormone (GH) synthesis were studied in monolayer cultures of rat pituitary tumor (GH₃) cells. PRL synthesis could not be inhibited by dopamine at concentrations ranging from 10⁻⁹ to 10⁻⁵ M. Bromocriptine inhibited PRL synthesis only at the very high concentration of 10⁻⁵ M, and this effect was not reversible by metoclopramide. GH synthesis was inhibited by 10⁻⁶ and 10⁻⁵ M dopamine but not by bromocriptine, and the dopamine effect was blocked by metoclopramide. These results suggest the existence of a defective dopaminergic regulation of PRL and GH synthesis in GH₃ cells.     

Muscarinic receptors on dopamine terminals in the cat caudate nucleus: Neuromodulation of [H]dopamine release in vitro by endogenous acetylcholine

The directly acting muscarinic receptor agonist oxotremorine (1.8–10 μM) produced an increase in electrically evoked [³H]dopamine release from slices of the cat caudate. The maximal increase caused by oxotremorine was 40%, and was antagonized by the muscarinic receptor blocking agent atropine (0.1 μM). Exposure to the acetylcholinesterase (AChE) inhibitor physostigmine (1 μM) resulted in a 50% increase in electrically evoked [³H]dopamine release. The increase caused by physostigmine was also antagonized by atropine (0.1 μM).Atropine did not, however, alter the modulations in [³H]dopamine release mediated by the dopamine autoreceptor: the increase in electrically evoked [³H]dopamine release caused by the dopamine receptor antagonist S-sulpiride (0.1 μM) and the decrease caused by the dopamine receptor agonist pergolide (30 nM) were unaffected by atropine (0.1 μM). These results indicate that the muscarinic receptor-mediated and dopamine autoreceptor-mediated presynaptic effects on [³H]dopamine release are independent.The present results suggest that in the electrically depolarized caudate slice in vitro, released endogenous acetylcholine may interact with muscarinic receptors faciliting depolarization-evoked [³H]dopamine release,ifAChE is inhibited. These muscarinic receptors may be located on dopamine nerve terminals. In the context of present neuroanatomical knowledge, the action of released endogenous acetylcholine on dopamine terminals may be a non-synaptic neuromodulation.     

β-adrenoceptor stimulation-induced increase in the number of α2-adrenoceptors of cerebral cortical membranes in rats

Effects of pre-treatment of synaptic membranes with β-adrenoceptor agonists and cholera toxin on [³H]clonidine and [³H]yohimbine binding were examined in rat cerebral cortex. Pre-incubation of cerebral cortical membranes with isoproterenol (10 or 200 μM) or dobutamine (1, 10 or 200 μM) at 37 °C for 40 min caused a significant elevation of specific [³H]clonidine binding but treatment with salbutamol (10 or 200 μM) did not. Scatchard analysis showed that 200 μM isoproterenol treatment resulted in a significant elevation of high affinity component of [³H]clonidine binding which was significantly decreased by the addition of 10μM GTP. A significant elevation in high affinity [³H]clonidine binding was observed by treatment with 100 μg/ml cholera toxin, while a significant decrease in low affinity one was by the treatment. Specific [³H]yohimbine binding was also elevated by 10 or 200 μM isoproterenol treatment. It is suggested that stimulation of β-receptors, presumably β₁-subtype could elevate the number of agonist and antagonist binding sites in α₂-receptors in synaptic membranes by partially mediated by stimulatory and/or inhibitory GTP binding regulatory proteins.     

KATP channel blockers selectively interact with A1-adenosine receptor mediated modulation of acetylcholine release in the rat hippocampus

In this study the role of ATP-sensitive K⁺ channels (K_ATP channels) in the A₁ receptor mediated presynaptic inhibitory modulation of acetylcholine release was investigated in the rat hippocampus. N⁶-Cyclohexyladenosine (CHA), the selective A₁-adenosine receptor agonist, reduced concentration-dependently the stimulation-evoked (2 Hz, 1 ms, 240 shocks) [³H]acetylcholine ([³H]ACh) release, from in vitro superfused hippocampal slices preloaded with [³H]choline, an effect prevented by the selective A₁ receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). By themselves, neither K_ATP channel openers, i.e. diazoxide, pinacidil and cromakalim, nor glibenclamide and glipizide, the inhibitors of K_ATP channels, exerted a significant effect on the resting and evoked release of [³H]ACh. Glibenclamide and glipizide (10–100 μM) completely prevented the inhibitory effect of 0.1 μM CHA and shifted the concentration response curve of CHA to the right. 4-Aminopyridine (10–100 μM), the non-selective potassium channel blocker, increased the evoked release of [³H]ACh, but in the presence of 4-aminopyridine, the inhibitory effect of CHA (0.1 μM) still persisted. Oxotremorine, the M₂ muscarinic receptor agonist, decreased the stimulation-evoked release of [³H]ACh, but its effect was not reversed by glibenclamide. 1,3-Diethyl-8-phenylxanthine (DPX), the selective A₁-antagonist, effectively displaced [³H]DPCPX in binding experiments, while in the case of glibenclamide and glipizide, only slight displacement was observed. In summary, our results suggest that K_ATP channels are functionally coupled to A₁ receptors present on cholinergic terminals of the hippocampus, and glibenclamide and glipizide, by interacting with K_ATP channels, relieve this inhibitory neuromodulation.     

Clorazepate synchronizes cultured rat C6 glioma in the early G1 phase of the cell cycle

A water soluble benzodiazepine, clorazepate, has been used to establish the point of benzodiazepine proliferative arrest in the rat C6 glioma. Clorazepate inhibited C6 proliferation in a dose-dependent manner with an IC₅₀ value of 280 μM, as judged by a nuclei counting procedure. Release of cells from a 48 h exposure to 350 μM clorazepate, at which over 70% of the cells were arrested, resulted in a synchronous entry into S phase 8–9 h later, as evidenced by a sharp increase in the incorporation of [³H]thymidine. This restriction point was demonstrated to be 2–3 h into the G₁ phase by measuring the length of G₁ in synchronized populations of C6 cells obtained by selection of mitotic figures from an asynchronous culture. Synchronous arrest of C6 by clorazepate required an exposure period of 24–36 h, approximately twice the doubling time of the cell line. A morphological study confirmed an early G₁ point of proliferative arrest. Clorazepate synchronized cells exhibited a uniform morphology with the majority of cells assuming a configuration representative of anchorage-dependent cells in an early phase of attachment. The majority of cells were somewhat rounded and attached to the substratum by cytoplasmic ‘skirts’ with punctate structures which may represent local adhesion points.     

Release of synaptosomal dopamine formed from tyrosine andl-dopa

The present report compares the synaptosomal release of [³H]dopamine, continuously forming from [³H]DOPA, with that of [¹⁴C]dopamine, forming from [¹⁴C]tyrosine as a marker of dopaminergic nerve endings. For the purpose of the comparison, synaptosomal (P₂) preparations from rat caudate nuclei were incubated withl-[³H]DOPA and [¹⁴C]tyrosine for 10 min and the particulates were rapidly separated from the medium postincubation. The separated fractions were analyzed for the level of double labelled (¹⁴C/³H) dopamine and the synaptosomal content of the labelled substrates. Of the total labelled dopamine formed, the fraction that was present in the medium, following the synaptosomal release, was determined. Tested were the release enhancing effects of various additions which included several known dopaminergic agents, serotonin and 5-hydroxytryptophan. The data show that the addition of dopamine to the incubation mixture to either 0.5 or 1.0 μM concentration markedly enhanced the release of labelled dopamine. Serotonin when added to 5.0 μM concentration also raised the medium content of labelled dopamine but it was ineffective at 1.0 μM. 5-Hydroxytryptophan, 1.0 or 5.0 μM, had no effect. For the comparison of the release enhancing effects of an addition of [¹⁴C]dopamine and [³H]dopamine, the corresponding release indices (release index = medium/total ratio of labelled dopamine in the presence of an addition divided by the same ratio in control (no addition) sample) were determined. The data indicate that the index of [¹⁴C]dopamine did not differ significantly from that of [³H]dopamine following the addition of either dopamine, serotonin or 5-hydroxytryptophan at any of the concentrations tested. A similar lack of difference between the index of [¹⁴C]dopamine and that of [³H]dopamine was observed following the addition of reserpine (1.8 μM), although a considerable enhancement of labelled dopamine release occurred. The addition of either amphetamine (9.1 μM) or amfonelic acid (9.1 μM) also enhanced labelled dopamine release but in their presence the index of [³H]dopamine was significantly higher than that of [¹⁴C]dopamine. Amfonelic acid preferentially raised the [³H]dopamine index at the lowest concentration of 0.91 μM that we have tested and also after only 5 min of incubation; coaddition of reserpine increased the [¹⁴C]dopamine release, thus abolishing the preferential effect of amfonelic acid on [³H]dopamine. When the incubation was performed without an addition (control sample), no difference (NS) was observed between the particulate to medium distribution of [¹⁴C]dopamine and that of [³H]dopamine after either 5, 10 or 15 min. The results suggest that (a) dopamine synaptosomally formed froml-DOPA may exist in a pool distinct from that dopamine arising from tyrosine hydroxylation and (b) the observed dopamine compartmentation may be due to some mechanism distinct from any possible participation of serotoninergic partivles. Also, the present results support the previously suggested existence of synaptosomal DOPA in pools.     

Effects of cholecystokinin tetrapeptide (CCK4) and anxiolytic drugs on the electrically evoked [H]5-hydroxytryptamine outflow from rat cortical slices

The outflow of [³H]5-hydroxytryptamine ([³H]5-HT) from electrically stimulated rat cortical slices was measured to ascertain the modulatory role of endogenous cholecystokinin (CCK) on the amine outflow and to test the hypothesis that different anxiolytic compounds inhibit 5-HT secretion. The [³H]5-HT outflow evoked at 10 Hz was increased up to +30% by CCK₄ 300–1000 nM, the effect being prevented by the CCK_B receptor antagonist GV 150013, 3 nM. The limited sensitivity to CCK₄ seemed to depend on 5-HT auto-receptor feedback because pre-treatment with 100 nM methiothepin enhanced the [³H]5-HT outflow and lowered the CCK₄ threshold concentration from 300 to 30 nM. In addition, pre-treatment with 1 μM bacitracin to inhibit CCK metabolism increased [³H]5-HT efflux. This effect was concentration-dependently counteracted by GV150013 suggesting the presence of an endogenous CCK positive modulation. GV150013 30 nM, the 5-HT_1A partial agonist buspirone 300 nM and the GABA_A receptor modulator diazepam 10 nM, known to have anxiolytic properties, all significantly reduced the [³H] amine outflow from cortical slices by about 20%. This inhibition depended on their interaction with their respective receptors, which seemed to restrain the activity of functionally interconnected glutamatergic interneurones. In fact, APV (50 μM) and NBQX (10 μM) prevented the effect of the anxiolytic compounds. Thus, anxiolytic drugs with different receptor targets can reduce 5-HT outflow by dampening the glutamatergic signal, and in turn, the secretory process of the serotonergic nerve ending.     

Deramciclane inhibits N-methyl-D-aspartate receptor function

Effects of the novel anxiolytic drug deramciclane on excitatory amino acid release and transmembrane Ca²⁺ ion flux processes were compared in rat cerebrocortical homogenates containing resealed plasmalemma fragments and nerve endings. Deramciclane (10 μM) significantly inhibited [³H]D-aspartate release and transmembrane Ca²⁺ flux to N-methyl-D-aspartate in the absence of Mg²⁺. By contrast, inhibition of [³H]D-aspartate release and transmembrane Ca²⁺ flux evoked by 0.1 mM (S)-α-amino-3-hydroxy-5-methyl-4-isoxazole propionate in the presence of Mg²⁺ and 10 μM cyclothiazide by 10 μM deramciclane was not significant. In the presence of N-methyl-D-aspartate receptor antagonists, deramciclane (10 μM) did not inhibit [³H]D-aspartate release to N-methyl-D-aspartate. These results suggest an involvement of the inhibition of a presynaptic N-methyl-D-aspartate receptor in the anxiolytic properties of deramciclane.     

Inhibition of NMDA-induced protein kinase C translocation by a Zn chelator: Implication of intracellular Zn

The role of intracellular Zn²⁺ in the translocation of protein kinase C from cytosol to membrane fractions was examined by the [³H]phorbol 12,13-dibutyrate (PDBu) binding method in guinea pig cerebral synaptoneurosomes. N-methyl-d-aspartate (NMDA, 100 μM) and calcium ionophore A23187 (0.3–30 μM) decreased the binding activity in the cytosol with a concomitant increase in the membrane fractions. Pretreatment of synaptoneurosomes with a heavy metal chelator, N,N,N′,N′-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN), inhibited the NMDA- and A23187-induced changes of the distribution of [³H]PDBu binding sites in cytosol and membrane fractions. The inhibitory effect of TPEN was negated by a preincubation of TPEN with equimolar Zn²⁺ but not by that with Ca²⁺. The addition of 500 μM Zn²⁺ to the lysate of synaptoneurosomes induced an increase of [³H]PDBu binding activity in the membrane fraction with a concomitant decrease in the cytosol fraction, as did 100 μM Ca²⁺. Low concentrations of Zn²⁺ (10 μM), which alone had no effect on the distribution of the binding, significantly enhanced the effect of 10 μM Ca²⁺ in the lysate. Under those conditions TPEN inhibited the Zn²⁺-potentiated Ca²⁺-dependent changes in the binding. These results suggest that intracellular Zn²⁺ is essential for the agonist-induced translocation of protein kinase C in guinea pig synaptoneurosomes.     

Ca mobilization and capacitative Ca entry regulate DNA synthesis in cultured chick retinal neuroepithelial cells

Release of Ca²⁺ from intracellular Ca²⁺ stores (Ca²⁺ mobilization) and capacitative Ca²⁺ entry have been shown to be inducible in neuroepithelial cells of the early embryonic chick retina. Both types of Ca²⁺ responses decline parallel with retinal progenitor cell proliferation. To investigate their potential role in the regulation of neuroepithelial cell proliferation, we studied the effects of 2,5-di-tert-butylhydroquinone (DBHQ), an inhibitor of the Ca²⁺ pump of intracellular Ca²⁺ stores, and of SK&F 96365, an inhibitor of capacitative Ca²⁺ entry, on DNA synthesis in retinal organ cultures from embryonic day 3 (E3) chicks and in dissociated cultures from E7 and E9 chick retinae. We demonstrate that both antagonists inhibit [³H]-thymidine incorporation in a dose-dependent manner without affecting cell viability or morphology. The inhibition of [³H]-thymidine incorporation by SK&F 96365 occurred in the same concentration range (IC₅₀: 4 μM) as the blockade of capacitative Ca²⁺ entry in the E3 retinal organ culture. At a concentration of 5 μM SK&F 96365, DNA synthesis was reduced by 71, 40 and 32% in the E3, E7 and E9 cultures, respectively. Application of DBHQ at concentrations which led to depletion of intracellular Ca²⁺ stores also inhibited [³H]-thymidine incorporation with IC₅₀ values of 20–30 μM in the different cultures. Our results suggest the involvement of Ca²⁺ mobilization and capacitative Ca²⁺ entry in the regulation of DNA synthesis in the developing neural retina.     

The interaction between β- and α2-adrenoceptors in cerebral cortical membranes: Isoproterenol-induced increase in [H]clonidine binding in rats

The pretreatment of rat cerebral cortical membranes with 10 or 100 μM isoproterenol at 37°C for 40 min caused a significant elevation of the B_max value of [³H]clonidine binding but pretreatment at 4°C did not affect the value. The isoproterenol-induced increase in the B_max value of the binding was higher in 50 mM Tris-HCl buffer (pH 7.7) than in Krebs-Ringer solution. In 50 mM Tris-HCl buffer (pH 7.7), treatment with isoproterenol reduced the B_max value of [³H]dihydroalprenolol binding but neitherK_d nor B_max of [³H]WB 4101 binding was affected by this treatment. Fitty μM propranolol or 100 μM GTP produced a significant reduction in isoproterenol-induced elevation of the B_max value of [³H]clonidine binding. In contrast, 100 μM cyclic AMP did not affect the control binding and 0.1 or 1 mM theophylline did not affect the isoproterenol-induced elevation of the binding. The only B_max value in high affinity binding of [³H]clonidine was increased by isoproterenol.It is suggested that isoproterenol increases the density of α₂-adrenoceptors in a temperature-dependent manner. The direct interaction between β- and α₂-receptor molecules and/or their indirect interaction, mediated by GTP regulatory proteins, would exist in the cerebral cortical membranes of rats.     

Interaction betweenα2- and β-adrenergic receptors in rat cerebral cortical membranes: clonidine-induced reduction in agonist and antagonist affinity for β-adrenergic receptors

The interaction betweenα₂- and β-adrenergic receptors was investigated in rat cerebral cortical membranes. Clonidine inhibition of [³H]dihydroalprenolol ([³H]DHA) binding resulted in biphasic competition curves with a mean Hill coefficient of 0.45. The addition of 1 μM yohimbine caused a rightward shift of the first portion of the clonidine inhibition curve. In the presence of 1 μM clonidine, the maximum concentration which did not inhibit [³H]DHA binding, inhibition curves of [³H]DHA binding by isoproterenol shifted to the right. A mean Hill coefficient increased from a control value of 0.63 to 0.76. Computer modeling analysis revealed that 1 μM clonidine decreased a β-adrenergic high-affinity state from 28% to 13%. However, the addition of 1 μM yohimbine completely prevented the clonidine-induced reduction in the β-adrenergic high-affinity state. In the presence of 200 μM GTP, the effect of clonidine was not observed. In addition,K_d andB_max values for[³H]p-aminoclonidine ([³H]PAC) binding were not significantly changed by the addition of 100 nM isoproterenol, the maximum concentration which did not inhibit [³H]PAC binding. Moreover, isoproterenol inhibition of [³H]PAC binding resulted in steep competition curves with a mean Hill coefficient of 0.97. The addition of 1 μM alprenolol did not affect the isoproterenol inhibition curve. These data demonstrated that clonidine caused a decrease in agonist and antagonist affinity for β-adrenergic receptors, while isoproterenol did not modulate the binding characteristics ofα₂-adrenergic receptors. Furthermore, these results suggest that regulation betweenα₂- and β-adrenergic receptors is not bidirectional, but is instead unidirectional fromα₂-adrenergic receptors to β-adrenergic receptors.     

Polyamine effects uponN-methyl-D-aspartate receptor functioning: differential alteration by glutamate and glycine site antagonists

Polyamines such as spermidine potentiate activation of theN-methyl-D-aspartate (NMDA)-type excitatory amino acid receptor. The goal of the present study was to investigate interactions between the putative polyamine binding site and previously described sites for glutamate and glycine. Binding of the high-potency PCP receptor ligand [³H]MK-801 to well-washed rat brain membranes was used as an in vitro probe of NMDA receptor activation. Spermidine concentration-response studies were performed in the absence and presence of both glutamate and glycine, with and withoutD-(−)-2-amino-5-phosphonovaleric acid (D(−)AP-5) or 7-chlorokynurenic acid (7Cl-KYN). Incubation in the presence of spermidine alone induced a 20.4-fold increase in [³H]MK-801 binding with an EC₅₀ value of 13.3 μM. The mean concentration of spermidine which induced maximal stimulation of binding was 130 μM (n = 10,S.E.M.= 24.66,range= 25–250 μM). Glutamate (10 μM) decreased the EC₅₀ value for spermidine-induced stimulation of [³H]MK-801 binding to 3.4 μM. Glycine (10 μM) did not significantly alter either maximum spermidine-induced [³H]MK-801 binding or the EC₅₀ value for spermidine-induced stimulation of [³H]MK-801 binding. Incubation in the presence of the specific glutamate antagonistD(−)AP-5 attenuated [³H]MK-801 binding in a glutamate-reversible fashion. The competitive glycine antagonist 7Cl-KYN decreased maximum spermidine-induced [³H]MK-801 binding in a glycine-reversible fashion. In addition, 7Cl-KYN increased the EC₅₀ value for spermidine-induced stimulation of [³H]MK-801 binding whileD(−)AP-5 was without effect. These findings suggest that glutamate and glycine regulate the polyamine binding site differentially. PCP-like agents induce a psychotomimetic state closely resembling schizophrenia by inhibiting NMDA receptor-mediated neurotransmission. The ability of polyamines to modulate NMDA receptor functioning suggests a potential site for pharmacological intervention.     

Downregulation of muscarinic- and 5-HT1B-mediated modulation of [H]acetylcholine release in hippocampal slices of rats with fimbria-fornix lesions and intrahippocampal grafts of septal origin

Adult Long-Evans female rats sustained electrolytic fimbria-fornix lesions and, two weeks later, received intrahippocampal suspension grafts of fetal septal tissue. Sham-operated and lesion-only rats served as controls. Between 6.5 and 8 months after grafting, both the [³H]choline accumulation and the electrically evoked [³H]acetylcholine ([³H]ACh) release were assessed in hippocampal slices. The release of [³H]ACh was measured in presence of atropine (muscarinic antagonist, 1 μM), physostigmine (acetylcholinesterase inhibitor, 0.1 μM), oxotremorine (muscarinic agonist, 0.01 μM–10 μM), mecamylamine (nicotinic antagonist, 10 μM), methiothepin (mixed 5-HT₁/5-HT₂ antagonist, 10 μM), 8-OH-DPAT (5-HT_1A agonist, 1 μM), 2-methyl-serotonin (5-HT₃ agonist, 1 μM) and CP 93129 (5-HT_1B agonist, 0.1 μM–100 μM), or without any drug application as a control. In lesion-only rats, the specific accumulation of [³H]choline was reduced to 46% of normal and the release of [³H]ACh to 32% (nCi) and 43% (% of tissue tritium content). In the grafted rats, these parameters were significantly increased to 63%, 98% and 116% of control, respectively. Physostigmine reduced the evoked [³H]ACh release and was significantly more effective in grafted (−70%) than in sham-operated (−56%) or lesion-only (−54%) rats. When physostigmine was superfused throughout, mecamylamine had no effect. Conversely, atropine induced a significant increase of [³H]ACh release in all groups, but this increase was significantly larger in sham-operated rats (+209%) than in the other groups (lesioned: +80%; grafted: +117%). Oxotremorine dose-dependently decreased the ([³H]ACh) release, but in lesion-only rats, this effect was significantly lower than in sham-operated rats. Whatever group was considered, 8-OH-DPAT, methiothepin and 2-methyl-serotonin failed to induce any significant effect on [³H]ACh release. In contrast, CP 93129 dose-dependently decreased [³H]ACh release. This effect was significantly weaker in grafted rats than in the rats of the two other groups. Our data confirm that cholinergic terminals in the intact hippocampus possess inhibitory muscarinic autoreceptors and serotonin heteroreceptors of the 5-HT_1B subtype. They also show that both types of receptors are still operative in the cholinergic terminals which survived the lesions and in the grafted cholinergic neurons. However, the muscarinic receptors in both lesioned and grafted rats, as well as the 5-HT_1B receptors in grafted rats show a sensitivity which seems to be downregulated in comparison to that found in sham-operated rats. In the grafted rats, both types of downregulations might contribute to (or reflect) an increased cholinergic function that results from a reduction of the inhibitory tonus which ACh and serotonin exert at the level of the cholinergic terminal.