The new compound GET73, N-[(4-trifluoromethyl)benzyl]4-methoxybutyramide, Regulates hippocampal Aminoacidergic transmission possibly via an allosteric modulation of mGlu5 receptor. Behavioural evidence of its "anti-alcohol" and anxiolytic properties.

The present article attempts to provide, on the basis of data emerging from studies carried out in our laboratories, a summary of the chemical and pharmacological properties of the new compound N-[(4-trifluoromethyl)benzyl]4- methoxybutyramide (GET73). Particular emphasis is given to findings obtained in vivo and in vitro suggesting that an allosteric modulation of metabotropic glutamate receptor 5 (mGlu5 receptor) by GET73 may represent the mechanism underlying the effects of the compound produced on rat hippocampal glutamate and GABA transmission. Furthermore, behavioural findings demonstrating how this new compound reduces alcohol intake, displays anxiolytic properties, and influences spatial memory in rats are also summarized. Since mGlu5 receptors play an important role in regulating several central actions of drugs of abuse, and the hippocampus is a crucial brain area involved in addiction, anxiety, and spatial memory, a possible link between mGlu5 receptor allosteric modulation and the profiles of action of GET73 is proposed, although to date no studies have yet explored GET73 binding at the mGlu5 receptor orthosteric and/or allosteric sites. Following a brief overview of glutamatergic neurotransmission, mGlu receptor structures and activation mechanisms, the general properties of mGlu5 receptor and its allosteric modulators are described in the first part of the review.

Adolescent exposure to nicotine and/or the cannabinoid agonist CP 55,940 induces gender-dependent long-lasting memory impairments and changes in brain nicotinic and CB(1) cannabinoid receptors.

We have analysed the long-term effects of adolescent (postnatal day 28-43) exposure of male and female rats to nicotine (NIC, 1.4 mg/kg/day) and/or the cannabinoid agonist CP 55,940 (CP, 0.4 mg/kg/day) on the following parameters measured in the adulthood: (1) the memory ability evaluated in the object location task (OL) and in the novel object test (NOT); (2) the anxiety-like behaviour in the elevated plus maze; and (3) nicotinic and CB(1) cannabinoid receptors in cingulated cortex and hippocampus. In the OL, all pharmacological treatments induced significant decreases in the DI of females, whereas no significant effects were found among males. In the NOT, NIC-treated females showed a significantly reduced DI, whereas the effect of the cannabinoid agonist (a decrease in the DI) was only significant in males. The anxiety-related behaviour was not changed by any drug. Both, nicotine and cannabinoid treatments induced a long-lasting increase in CB(1) receptor activity (CP-stimulated GTPγS binding) in male rats, and the nicotine treatment also induced a decrease in nicotinic receptor density in the prefrontal cortex of females. The results show gender-dependent harmful effects of both drugs and long-lasting changes in CB(1) and nicotinic receptors.

Cannabinoid CB1 receptors in the paraventricular nucleus and central control of penile erection: immunocytochemistry, autoradiography and behavioral studies.

[N-(piperidin-1-yl)-5-(4-chlorophenyl)-4-methyl-1H-pyrazole-3-carboxyamide] (SR 141716A), a selective cannabinoid CB1 receptor antagonist, injected into the paraventricular nucleus of the hypothalamus (PVN) of male rats, induces penile erection. This effect is mediated by the release of glutamic acid, which in turn activates central oxytocinergic neurons mediating penile erection. Double immunofluorescence studies with selective antibodies against CB1 receptors, glutamic acid transporters (vesicular glutamate transporters 1 and 2 (VGlut1 and VGlut2), glutamic acid decarboxylase-67 (GAD67) and oxytocin itself, have shown that CB1 receptors in the PVN are located mainly in GABAergic terminals and fibers surrounding oxytocinergic cell bodies. As GABAergic synapses in the PVN impinge directly on oxytocinergic neurons or on excitatory glutamatergic synapses, which also impinge on oxytocinergic neurons, these results suggest that the blockade of CB1 receptors decreases GABA release in the PVN, increasing in turn glutamatergic neurotransmission to activate oxytocinergic neurons mediating penile erection. Autoradiography studies with [(3)H](-)-CP 55,940 show that chronic treatment with SR 141716A for 15 days twice daily (1 mg/kg i.p.) significantly increases the density of CB1 receptors in the PVN. This increase occurs concomitantly with an almost twofold increase in the pro-erectile effect of SR 141716A injected into the PVN as compared with control rats. The present findings confirm that PVN CB1 receptors, localized mainly in GABAergic synapses that control in an inhibitory fashion excitatory synapses, exert an inhibitory control on penile erection, demonstrating for the first time that chronic blockade of CB1 receptors by SR 141716A increases the density of these receptors in the PVN. This increase is related to an enhanced pro-erectile effect of SR 141716A, which is still present 3 days after the end of the chronic treatment.

(-)S amisulpride binds with high affinity to cloned dopamine D(3) and D(2) receptors.

Amisulpride is a substituted benzamide antipsychotic with nanomolar affinity and high selectivity for dopamine D(2) and dopamine D(3) receptors. The interaction of racemic (+/-)RS amisulpride and its two enantiomers (+)R and (-)S with dopamine D(2) and dopamine D(3) receptors subtypes were compared with that of haloperidol. Binding studies were performed using either [3H]spiperone or [3H]nemonapride in baculovirus/Spodoptera frugiperda insect (Sf-9) cell system expressing either the human dopamine recombinant D(2)long (hD(2L)) or the rat dopamine recombinant D(3) (rD(3)) receptors. K(i) values at dopamine rD(3) receptors were similar regardless of the radioligand used, whereas at hD(2L) receptors values were higher using [3H]spiperone than [3H]nemonapride. However, the rank order of compound potency against radiolabeled spiperone or nemonapride both at dopamine hD(2L) and at dopamine rD(3) receptors was similar. (-)S amisulpride displaced [3H]spiperone or [3H]nemonapride binding from both dopamine hD(2L) or dopamine rD(3) receptors, being twofold more potent than the racemic form and 38-19-fold more potent than (+)R enantiomer. Both racemic and the (-)S enantiomer exhibited 2-4 ([3H]spiperone)- and 3-4 ([3H]nemonapride)-fold higher affinity than haloperidol for dopamine rD(3) receptor, respectively. The (+)R enantiomer has weaker affinity with respect to haloperidol for both dopamine hD(2L) and dopamine rD(3) receptors. Our results show that (-)S amisulpride is the active enantiomer of amisulpride, showing high affinity for dopamine D(3) and dopamine D(2) receptors.

Quantitative autoradiographic distribution of gamma-hydroxybutyric acid binding sites in human and monkey brain.

gamma-Hydroxybutyric acid (GHB), a naturally occurring metabolite of GABA, is present in micromolar concentrations in various areas of the mammalian brain. Specific GHB binding sites, uptake system, synthetic and metabolizing enzymes have been identified in CNS. The present study shows the anatomical distribution of GHB binding sites in sections of primate (squirrel monkey) and human brain by radioligand quantitative autoradiography. In both species the highest densities of binding sites were found in the hippocampus, high to moderate densities in cortical areas (frontal, temporal, insular, cingulate and entorhinal) and low densities in the striatum; no binding sites were detected in the cerebellum. High density of GHB binding was found in the monkey amygdala. In addition the binding characteristics of [(3)H]GHB to membrane preparations of human brain cortex were examined. Scatchard analysis and saturation curves revealed both a high (K(d1) 92+/-4.4 nM; B(max1) 1027+/-110 fmol/mg protein) and a low-affinity binding site (K(d2) 916+/-42 nM; B(max2) 8770+/-159 fmol/mg protein). The present study is the first report on the autoradiographic distribution of specific GHB binding sites in the primate and human brain: such distribution is in both species in good agreement with the distribution found in the rat brain.

Reconstitution of the human 5-HT(1D) receptor-G-protein coupling: evidence for constitutive activity and multiple receptor conformations.

The 5-hydroxytryptamine (5-HT) 1D/1B receptors have gained particular interest as potential targets for treatment of migraine and depression. G-protein coupling and other intrinsic properties of the human 5-HT(1D) receptor were studied using a baculovirus-based expression system in Sf9 cells. Coexpression of the human 5-HT(1D) receptor with Galpha(i1), alpha(i2), alpha(i3), or Galpha(o)-proteins and Gbeta(1)gamma(2)-subunits reconstituted a Gpp(NH)p-sensitive, high affinity binding of [(3)H]5-HT to this receptor, whereas the Galpha(q)beta(1)gamma(2) heterotrimer was ineffective in this respect. Competition of [(3)H]5-HT binding by various compounds confirmed that coexpression of the human 5-HT(1D) receptor with Galpha(i/o)beta(1)gamma(2) reconstitutes the receptor in a high affinity agonist binding state, having the same pharmacological profile as the receptor expressed in mammalian cells. Binding of the antagonist ocaperidone to the human 5-HT(1D) receptor in coupled or noncoupled state was analyzed. This compound competed with [(3)H]5-HT binding more potently on the human 5-HT(1D) receptor in the noncoupled state, showing its inverse agonistic character. Ocaperidone acted as a competitive inhibitor of [(3)H]5-HT binding when tested with the coupled receptor form but not so when tested with the noncoupled receptor preparation. Finally, [(35)S]GTPgammaS binding experiments using the inverse agonist ocaperidone revealed a high level of constitutive activity of the human 5-HT(1D) receptor. Taken together, the reconstitution of the human 5-HT(1D) receptor-G-protein coupling using baculovirus-infected Sf9 cells made possible the assessment of coupling specificity and the detection of different binding states of the receptor induced by G-protein coupling or ligand binding.

Distribution of GABA(B) receptor mRNAs in the rat brain and peripheral organs.

GABA, the predominant inhibitory neurotransmitter present in the mammalian CNS, is also found in the periphery. GABA actions are mediated by the ionotropic GABA(A)/GABA(C) receptors, as well as the metabotropic GABA(B) receptor. The rat GABA(B) receptor has recently been cloned and two cDNA clones have been isolated encoding two isoforms of the receptor, GABA(B)R1a and R1b. Northern blot analysis revealed the presence of both transcripts in the rat brain using specific cDNA probes for GABA(B)R1a and R1b, respectively. However, Northern blot analysis, hybridized with a probe containing a sequence common to both isoforms, revealed specific RNAs in the rat brain and in testis, but not in other peripheral tissues. In the present study, by using the more sensitive reverse transcriptase-polymerase chain reaction with a specific set of primers for each isoform and Southern blot analysis, we found that both isoforms of the GABA(B) receptor are expressed not only throughout the brain but also in all peripheral organs examined, including heart, spleen, lung, liver, small intestine, large intestine, kidney, stomach, adrenal, testis, ovary and urinary bladder. The peripheral distribution of GABA(B)R1 mRNAs supports the notion of a physiological role for GABA in the control of a wide range of peripheral organs.

Chronic morphine and naltrexone fail to modify mu-opioid receptor mRNA levels in the rat brain.

Previous radioligand-binding studies have reported conflicting results concerning the effect of chronic morphine administration on the regulation of mu-opioid receptor (MOR) density. On the other hand, chronic administration of an opioid antagonist, such as naltrexone, has been shown to increase the density of the MOR. In order to determine if the changes in the MOR are associated with alterations in receptor mRNA levels, we investigated MOR gene expression following chronic treatment with morphine and/or naltrexone. MOR mRNA levels, determined by the ribonuclease protection assay (RPA), were unchanged with respect to control during chronic morphine treatment and morphine withdrawal in each of the analysed brain areas. Furthermore, chronic administration of naltrexone did not result in changes of MOR mRNA levels in rat striatum of naive and morphine-dependent rats, suggesting that the up-regulation of the MOR density, at least in this tissue, is not regulated at transcriptional level.

Thyroid transcription factor 1 activates the promoter of the thyrotropin receptor gene.

The TSH receptor (TSHr) is one of the most important thyroid differentiation markers. The binding of the TSH hormone to its receptor is an essential step in the modulation of thyroid function and differentiation. Here we report that the thyroid transcription factor 1 (TTF1), a transcription factor essential for thyroid-specific gene expression, binds to the TSHr minimal promoter. The promoter, when mutated at this binding site, shows a decreased activity in thyroid cells. In cotransfection experiments in nonthyroid cells, TTF1 is able to trans-activate the TSHr minimal promoter. This finding strengthens the importance of TTF1 in the maintenance of thyroid differentiation. The promoters of the main thyroid differentiation markers thyroglobulin, thyroperoxidase, and now TSHr, are regulated by TTF1.

Cathepsin G--induced release of PAI-1 in the culture medium of endothelial cells: a new thrombogenic role for polymorphonuclear leukocytes?

Activated polymorphonuclear leukocytes (PMNs) may affect the integrity of blood vessels by endothelial cell injury. We investigated the effects of cathepsin G purified from human neutrophils on the fibrinolytic potential of cultured human umbilical vein endothelial cells (HUVECs). Cathepsin G (5 and 10 micrograms/ml) induced marked intercellular gap formation after 1 hour of treatment, whereas 1 microgram/ml did not, even after 6 hours incubation. In contrast, plasminogen activator inhibitor-1 (PAI-1) antigen levels, measured by a double antibody enzyme-linked immunosorbent assay, were significantly increased in culture media (CM) on cathepsin G (1 microgram/ml) treatment after 15 minutes (5.1 +/- 1.2 ng/ml vs 2.6 +/- 0.6 ng/ml for controls, p < 0.01) and 6 hours of incubation (69.6 +/- 17.5 ng/ml vs 40.0 +/- 9.0 ng/ml for controls, p < 0.01). Likewise, PAI activity, measured by reverse fibrin autography, increased on cell treatment with cathepsin G. Preincubation of cathepsin G with eglin C (10 micrograms/ml) almost completely abolished the increase in both PAI antigen and activity levels induced by cathepsin G. Cycloheximide, a protein synthesis inhibitor, did not block cathepsin G-induced PAI-1 release. PAI-1 mRNA levels were not affected by HUVEC treatment with cathepsin G (1 microgram/ml for 15 minutes), even after 24 hours. In the extracellular matrix (ECM) PAI-1 antigen levels decreased to 77% and 40% of controls, respectively, after 15 minutes and 6 hours of cathepsin G (1 micrograms/ml) treatment. Reverse fibrin autography also demonstrated a dose-dependent reduction of PAI activity in the ECM on 6 hours of cell treatment with 1 or 5 micrograms/ml cathepsin G. Moreover, ECM prepared from confluent HUVECs released PAI-1 in supernatants on 1 micrograms/ml cathepsin G incubation in a cell-free system. Tissue-type plasminogen activator (t-PA) activity was strongly depressed on cathepsin G treatment, both in CM from HUVECs or in a cell-free system. Finally, PAI-1 was also released from cathepsin G-stimulated platelets in a dose-dependent manner. In summary, our results support a potentially thrombogenic role of cathepsin G, which could impair the fibrinolytic potential of the endothelium. These data give a new insight into the mechanisms by which activated PMNs may promote thrombus formation. On the other hand, the decrease of PAI-1 in ECM could favor penetration and migration of inflammatory or tumor cells through the subendothelial layers.

Protective, restorative, and therapeutic properties of recombinant human IL-1 in rodent models.

Human rIL-1 alpha and -1 beta are shown to increase significantly the CFU-culture activity in the spleen as well as at other sites after i.v. or i.p. administration. IL-1 can also significantly increase survival and can "rescue" a number of animals if administered either before or after lethal doses of cyclophosphamide or gamma-irradiation. The protective and reconstitutive activities of the rIL-1 are shown to correlate with increased CFU-culture frequency and total number, as well as increased cellularity in the bone marrow and peripheral blood, suggesting that this is one of their mechanisms of action. The sequence and timing of administration of human rIL-1 is critical for the protection or rescue of animals receiving DNA-damaging agents; maximal activity is achieved when IL-1 is given 20 h before insult or 48 h after alkylating agent administration. Minimal therapeutic activity is observed with IL-1 as a single agent for the treatment of metastatic disease compared with other biologic response modifiers including IFN-gamma.

Continuous subcutaneous insulin infusion (CSII) in pregnant diabetic patients.

The efficacy of the insulin infusion pump (CSII) in pregnancy was examined in 12 diabetic patients and compared with intermittent insulin therapy (IIT). In patients poorly controlled on IIT constant and rapid equilibrium was achieved with CSII (mean of glucose levels: CSII versus IIT = 84 versus 137 mg/dl; S.D. = 36 versus 63 mg/dl; mean amplitude of glycemic excursion (MAGE) = 65 versus 112 mg/dl. In patients well controlled on IIT, CSII led to a reduction in the variation of glucose excursions (S.D. = 29 versus 36 mg/dl; MAGE = 48 versus 76 mg/dl). CSII generally produced a reduction of 20-37 per cent of daily insulin dose (in three cases there was an increase of dose with the achievement of glycemic control). Furthermore in CSII treated-patients amniotic glucose, insulin and C-peptide concentrations were found to be in the normal range (22.1 +/- 10.1 mg/dl; 5.2 +/- 2.7 microU/ml; 1.25 +/- 0.71 ng/ml, respectively). All infants were born at or near-term, had no macrosomia or neonatal problems. It is concluded that CSII is a highly efficient way to achieve normal glucose levels in pregnancy, not only in type I, but also in type II or gestational diabetes.