Bone-targeted Src SH2 inhibitors block Src cellular activity and osteoclast-mediated resorption.

Src, a nonreceptor tyrosine kinase, is an important regulator of osteoclast-mediated resorption. We have investigated whether compounds that bind to the Src SH2 domain inhibit Src activity in cells and decrease osteoclast-mediated resorption. Compounds were examined for binding to the Src SH2 domain in vitro using a fluorescence polarization binding assay. Experiments were carried out with compounds demonstrating in vitro binding activity (nmol/L range) to determine if they inhibit Src SH2 binding and Src function in cells, demonstrate blockade of Src signaling, and lack cellular toxicity. Cell-based assays included: (1) a mammalian two-hybrid assay; (2) morphological reversion and growth inhibition of cSrcY527F-transformed cells; and (3) inhibition of cortactin phosphorylation in csk-/- cells. The Src SH2 binding compounds inhibit Src activity in all three of these mechanism-based assays. The compounds described were synthesized to contain nonhydrolyzable phosphotyrosine mimics that bind to bone. These compounds were further tested and found to inhibit rabbit osteoclast-mediated resorption of dentine. These results indicate that compounds that bind to the Src SH2 domain can inhibit Src activity in cells and inhibit osteoclast-mediated resorption.

Cbl associates with Pyk2 and Src to regulate Src kinase activity, alpha(v)beta(3) integrin-mediated signaling, cell adhesion, and osteoclast motility.

The signaling events downstream of integrins that regulate cell attachment and motility are only partially understood. Using osteoclasts and transfected 293 cells, we find that a molecular complex comprising Src, Pyk2, and Cbl functions to regulate cell adhesion and motility. The activation of integrin alpha(v)beta(3) induces the [Ca(2+)](i)-dependent phosphorylation of Pyk2 Y402, its association with Src SH2, Src activation, and the Src SH3-dependent recruitment and phosphorylation of c-Cbl. Furthermore, the PTB domain of Cbl is shown to bind to phosphorylated Tyr-416 in the activation loop of Src, the autophosphorylation site of Src, inhibiting Src kinase activity and integrin-mediated adhesion. Finally, we show that deletion of c Src or c-Cbl leads to a decrease in osteoclast migration. Thus, binding of alpha(v)beta(3) integrin induces the formation of a Pyk2/Src/Cbl complex in which Cbl is a key regulator of Src kinase activity and of cell adhesion and migration. These findings may explain the osteopetrotic phenotype in the Src(-/-) mice.

Effects of RU 52583, an alpha 2-antagonist, on memory in rats with excitotoxic damage to the septal area.

The anti-amnesic action of RU 52583, an alpha 2-adrenergic receptor antagonist, was evaluated through performance of spatial tasks in a radial maze by rats with N-methyl-D-aspartic acid (NMDA) lesion of the medial septal (MS) nuclei. Memory performance of lesioned or sham-operated rats was evaluated by measuring reference memory as long-term maintenance of an acquired performance and working memory or memory for recent events. The lesion: a produced significant impairments of the animals' memory performance, b) significantly reduced the sodium-dependent high-affinity choline uptake in the hippocampal formation, and c) deeply disrupted cholinergic hippocampal theta waves. Oral administration of RU 52583 at 1 and 2 mg/kg (tested doses: 1-5 mg/kg) prior to performance of the task markedly reduced memory impairments, whereas idazoxan, another alpha 2-adrenergic receptor antagonist, had no effect at tested doses (2-5 mg/kg). Cholinergic drugs--arecoline at 0.1 and 1 mg/kg (tested doses: 0.05-1 mg/kg) and physostigmine at 0.02 and 0.1 mg/kg (tested doses: 1, 2, and 5 mg/kg)-administered intraperitoneally showed a tendency to alleviate memory deficits. The present results show that the alpha 2-adrenergic antagonist RU 52583 possesses cognition-enhancing properties in rats with damage to the septohippocampal system.

Roman strains as a psychogenetic model for the study of working memory: behavioral and biochemical data.

Performances of male rats of the Roman High- (RHA), Roman Control- (RCA) and Roman Low- (RLA) Avoidance strains were compared in two working memory tests, a spatial one, the radial maze, and a nonspatial one, an object recognition test. The same rats were subjected to measures of emotional reactivity and of different forms of motor activity and finally to measures of cholinergic and aminergic activities in the hippocampus, frontal cortex and striatum. Compared to RHA, RLA performed better in the two working memory tests, displayed "anxiety" and had also lower levels of exploratory locomotor activity. Hippocampal ChAT activity was higher in RLA than in RHA. Levels of DA and DOPAC in the striatum were higher in RLA compared to RHA, whereas in the frontal cortex they were lower. For most of these measures, RCA were intermediate between RLA and RHA. These results confirm and extend the finding that the Roman strains are not only a genetic model for two-way avoidance conditioning but also for working memory.

Angiotensin-converting enzyme in rat brain and extraneural tissues visualized by quantitative autoradiography using 3H-trandolaprilate.

Tritium-labeled trandolaprilate (RU 44403), the active diacid form of the potent and long-acting angiotensin-converting enzyme (ACE) inhibitor trandolapril, has been evaluated as a new autoradiographic marker for the enzyme. The characteristics of 3H-trandolaprilate binding were first determined autoradiographically in tissue sections from rat brain (caudate-putamen) and kidney. 3H-Trandolaprilate binds saturably to these tissues, and with very high affinity (Kd values, 0.36 and 0.13 nM, respectively), and appears to show good selectivity for the enzyme. Due to its high affinity (approximately 100 times that of captopril), the performance of 3H-trandolaprilate as an autoradiographic marker is comparable to that of the recently described 125I-labeled derivative of lisinopril (125I-351A). Saturation and displacement studies in serial sections from a variety of central and peripheral tissues confirmed that the specificity of labeling was similar throughout. The anatomical distribution of ACE visualized with 3H-trandolaprilate in these tissues was close to that described for 3H-captopril and 125I-351A with some minor differences which might arise from difference in the specificity of the ligands.

Benzodiazepine binding sites in the cingulate cortex after lesion of the noradrenaline and dopamine containing afferents.

The distribution of benzodiazepine binding sites was analysed in the cingulate cortex of the rat brain by quantitative radioautography of brain sections incubated with a full agonist benzodiazepine ligand, 3H-flunitrazepam (3H-FLU), or with a partial agonist with non benzodiazepine structure, (7-3H)-4hydroxy-N(4,5-dihydroxy-2-thiazolyl)-6 methoxy-3-quinoline (3H-RU 43028), after lesion of noradrenaline (NA) and dopamine (DA) containing afferents to this structure. NA denervation was obtained by systemic administration of N-(2-chlorethyl)-N ethyl-2-bromobenzylamine (DSP4) and destruction of both NA and DA containing afferents was induced by unilateral injection of 6-hydroxydopamine (6-OHDA) in the middle forebrain bundle (MFB). A similar caudo-rostral pattern of distribution was found in the cingulate cortex after incubation with these two ligands which bound a greater number of sites in the anterior portion of the structure. In spite of a very precise anatomical sampling (200 micron intervals along the postero-anterior axis) no significant difference was observed when intact and lesioned brains were compared. It is concluded that benzodiazepine binding sites eventually localized on catecholaminergic afferents to the cingulate cortex do not represent a significant proportion of the total population of these sites in this structure.

Tolerance to the serotonin 5-HT1 agonist RU 24969 and effects on dopaminergic behaviour.

The pharmacological responses to intraperitoneal injection of the serotonin (5-HT) 5-HT1 agonist RU 24969 (0.25-5 mg/kg) were studied in rats either after single administrations or after repeated treatment (5 mg/kg per day for 3 days). The following effects were recorded after a single dose: (A) a strong increase in locomotor activity in intact rats and its potentiation after 5,7-dihydroxytryptamine lesion of 5-HT neurons; (B) at a low dose, a potent enhancement of the circling behaviour induced by the dopamine (DA) D2 agonist LY 171555 in 6-hydroxydopamine-lesioned rats; (C) an early reduction (2 h) of 5-hydroxyindole acetic acid levels in the striatum and the nucleus accumbens followed by a late increase (24 h) in the latter structure. The following modifications were observed 24 h after the repeated treatment with RU 24969: (A) the locomotor effect of the drug was strikingly reduced both in intact and 5,7-dihydroxytryptamine-lesioned animals. On the contrary, the locomotion elicited by the DA releaser d-amphetamine, or the 5-HT1A agonist 8-OH-DPAT, was unchanged; (B) the rotation scores of 6-hydroxydopamine-lesioned rats injected with LY 171555 after a low dose of RU 24969, were greatly reduced. Moreover, the circling response was almost abolished in rats treated with the DA agonist alone; (C) the early reduction of 5-hydroxyindole acetic acid levels was antagonized while the late increase was enhanced. It is concluded that both the state of tolerance and the reversal of the action of RU 24969 that followed repeated treatment might be related to down-regulation of a subtype of the 5-HT1 receptor, possibly the 5-HT1B subtype, that would play a critical role in the expression of DA-mediated behaviour, locomotor activity and 5-HT metabolism.

Catecholamine metabolism in the rat locus coeruleus as studied by in vivo differential pulse voltammetry. III. Evidence for the existence of an alpha 2-adrenergic tonic inhibition in behaving rats.

One of the various regulations controlling the noradrenergic (NA) locus coeruleus (LC) activity has been proved to be alpha 2 adrenergic specific, on the basis of electrophysiological data obtained in anesthetized preparations. To assess, under rigorously chronic conditions, the existence of such an inhibition, recordings of LC catechol metabolic activity were performed with in vivo differential pulse voltammetry. A guiding cannula and appropriate wires were implanted under anesthesia. After 48 h of recovery a carbon fiber electrode was threaded to the LC through this cannula to monitor the LC catechol oxidation current. Piperoxane 60 mg/kg i.p. and yohimbine 10 mg/kg i.p. induced an increase in catechol oxidation current to approximately 300% of baseline (100%) values. Graded doses of piperoxane (1-100 mg/kg i.p.) induced a dose dependent increase in LC catechol metabolic activity (ED50 = 29.7 mg/kg). These changes in catechol oxidation current were confirmed either by combined electrophysiological and electrochemical recordings in the LC of an anesthetized preparation, or by postmortem HPLC catechol determinations on LC microdissections. By contrast, guanfacine 1 mg/kg and clonidine (10-200 micrograms/kg i.p.) induced a dose dependent decrease in catechol peak height. Clonidine 50 micrograms/kg reversed the effect of piperoxane 30 mg/kg i.p. On the other hand, a highly selective alpha 1 antagonist, such as prazosin (1 mg/kg i.p.), evoked only a small increase in catechol peak (11% above saline effect). This data is consistent with previously reported electrophysiological, biochemical and autoradiographic data. They confirm the presence of a tonic alpha 2 adrenergic inhibition on NA-LC cell activity, in behaving rats.

Lesion of noradrenergic neurones with DSP4 does not modify benzodiazepine receptor binding in cortex and hippocampus of rat.

The effect of lesioning noradrenergic pathways on the benzodiazepine receptor has been studied using a novel neurotoxic agent, N(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) which has better selectivity than the classical 6-hydroxydopamine (6-OHDA) towards noradrenergic neurones, and which has the added advantage of being injected systemically rather than intracerebrally. Three different radioligands were used: an agonist, [3H]flunitrazepam, an antagonist, [3H]Ro 15-1788, and a partial agonist [3H]RU 43028. Binding was measured using membrane homogenates from the cortex and hippocampus of the rat, two regions of the brain which receive an extensive noradrenergic innervation. In contrast to previous reports of a decrease in the binding of [3H]flunitrazepam following lesioning with 6-OHDA, no significant change was observed in either the affinity (KD) or the number of sites (Bmax) of any one of these ligands after lesioning with DSP4. While the reasons for this discrepancy are not clear, these results do not confirm that destruction of noradrenergic afferents to the cortex or hippocampus leads to any modification of the benzodiazepine receptor as demonstrated by ligand binding.