Rosanna Di Toro

Nociceptin-induced internalization of the ORL1 receptor in human neuroblastoma cells.

Nociceptin/orphanin FQ (NC) has been proposed as endogenous ligand of the opioid receptor-like 1 (ORL1) receptor. We investigated NC-induced internalization and recycling of the ORL1 receptor in SK-N-BE human neuroblastoma cells. Internalization was proven by receptor binding assay on viable cells. NC promotes a time- and concentration-dependent internalization of the ORL1 receptor (57% of cell surface receptors are lost after 30 min exposure to 1 μM NC) in a clathrin- and ATP- dependent manner. After 30 min exposure to NC, ORL1 receptor internalization is partially reversible and recycling is dependent on acid phosphatases. Over-expression of β-arrestin 2 increases NC-promoted internalization of the ORL1 receptor. These events contribute to NC signaling in neuronal cells through sequestration and recycling of the ORL1 receptor.

Effects of specific bile acids on c-fos messenger RNA levels in human colon carcinoma Caco-2 cells

Bile acids may play a role in the pathogenesis of intestinal inflammation by activating the signalling pathways that control cell proliferation, among other cell systems. We investigated the action of different bile acids, particularly chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA), on steady-state and transcriptional regulation of the protooncogene c-fos, involved in the regulation of cell proliferation and differentiation, in colon carcinoma Caco-2 cells. Specific bile acids had a stimulatory effect of on the expression of c-fos mRNA. This proved to be concentration- and time-dependent and may be partly due to an increase in the rate of transcription of the corresponding gene rather than to any change in the stability of mRNA. In Caco-2 cells exposed to 250 microM CDCA for 1 h a maximal increase of c-fos mRNA ( approximately 2.5-fold induction over the control) was observed; deoxycholic acid (DCA; 250 microM) and lithocholic acid (LCA; 250 microM) were less effective (approximately 2-fold induction over the control). UDCA and cholic acid (CA) did not modify c-fos gene expression in this cell line. Finally, we investigated the role of protein kinase C (PKC) in transcriptional regulation of the c-fos gene by bile acids. Although induction of c-fos by 12-O-tetradecanoyl 13-acetate (10 nM), a potent PKC activator, was completely antagonised by bis-indolyl-maleimide I (1 microM); only about 40% of the bile acid-mediated rise in c-fos mRNA was blocked. Thus it appears that PKC, as well as other signalling pathways, is involved in CDCA-, DCA- and LCA-induced c-fos gene expression.

Substituted 1-phenyl-2-cyclopropylmethylamines with high affinity and selectivity for sigma sites

A series of 1-phenyl-2-cyclopropylmethylamines structurally related to (+)- and (-)-MPCB were synthesized and their binding affinities for sigma1, sigma2, opioid and dopamine (D2) receptors were evaluated. Substitution of the cis-N-normetazocine with different aminic moieties provided compounds with high affinity and selectivity for sigma binding sites with respect to opioid and dopamine (D2) receptors. The observed increase in sigma2 affinity as compared to the parent (+)-MPCB, supports the idea that the particular stereochemistry of (+)-cis-N-normetazocine affects sigma1 selectivity but does not affect sigma1 affinity. The (+/-)-cis isomers of methyl 2-[(1-adamantylamino)methyl]-1-phenylcyclopropane-1-carboxyl ate (18) displayed a higher affinity and selectivity for the sigma1 and sigma2 receptor subtypes compared to the (+/-)-trans 19. Interestingly, the enantiomer (-)-cis 18 displayed a preference for sigma1 receptor subtype whereas the (+)-cis 18 did for sigma2. These results prompt us to synthesize compounds with modification of nitrogen and carboxyl groups. The compounds obtained showed high affinities and selectivity for sigma sites. Moreover, modifications of carboxyl groups provided compounds with the highest affinities in the series. In particular, compound 25 with reverse-type ester showed a Ki of 0.6 and 4.05 nM for sigma1 and sigma2 binding sites, respectively.

Expression of the repressor element‐1 silencing transcription factor (REST) is influenced by insulin‐like growth factor‐I in differentiating human neuroblastoma cells

The repressor element‐1 (RE‐1) silencing transcription factor (REST) interacts with an RE‐1 cis element and represses the transcription of neuron‐specific genes in neuronal progenitors but is down‐regulated in post‐mitotic neurons. We report that REST expression is modified, in a time‐dependent manner, in SH‐SY5Y neuroblastoma cells exposed to insulin‐like growth factor I (IGF‐I), a polypeptide hormone affecting various aspects of neuronal induction and maturation. REST is increased in cells treated with IGF‐I for 2 days and then declines in 5‐day‐treated cells concomitant with a progressive neurite extension. To investigate any role played by REST in neurodifferentiation by IGF‐I, we employed an antisense oligonucleotide (AS‐ODN) complementary to REST mRNA. In AS‐ODN‐treated cells, the effects elicited by IGF‐I on cell proliferation are not influenced whereas a marked decrease of REST significantly increases neurite elongation without any gross perturbation of neurogenesis. Synapsin I and βIII‐tubulin gene promoters contain an RE‐1 motif and their transcription is repressed by REST; both of them are increased in cells exposed to IGF‐I for 5 days and further elevated by AS‐ODN treatment. A parallel increase of growth cone‐associated protein 43, a protein chosen as a neuronal marker not directly regulated by REST, is also observed. Therefore, REST is elevated during early steps of neural induction by IGF‐I and could contribute to down‐regulate genes not yet required by the differentiation program while it declines later for the acquisition of neural phenotypes. These results suggest a model in which differentiating neuroblastoma cells determine their extent of neurite outgrowth on the basis of REST disappearance.

Opioid peptide response to spinal cord stimulation in chronic critical limb ischemia

Twelve patients with chronic critical limb ischemia in whom a spinal cord stimulation (SCS) system had been implanted for at least one year had increased microvascular flow and achieved healing of trophic acral lesions. After switching off the system, the clinical improvement persisted for 10 days and the neurohormonal pattern showed high plasma values of beta-endorphin and Met-enkephalin, normal dynorphin B, endothelin-1 and catecholamines, and low nitric oxide. Met-enkephalin levels were further increased (P < 0.01) immediately after switching on the electrical stimulation again. The persistence of high plasma opioid levels after switching off the spinal cord stimulation explains the absence of subjective complaints and suggests an involvement of opioids in the regulation and improvement of the microcirculation.

Regulation of δ opioid receptors by Δ9-tetrahydrocannabinol in NG108-15 hybrid cells

Abstract In this study we employed the neuroblastoma x glioma NG 108-15 cell line as a model for investigating the effects of long-term activation of cannabinoid receptors on δ opioid receptor desensitization, down-regulation and gene expression. Exposure of NG 108-15 cells to (−)- Δ 9 -tetrahydrocannabinol ( Δ 9 -THC) reduced opioid receptor binding, evaluated in intact cells, by ≈ 40 – 45% in cells exposed for 24 h to 50 and 100 nM Δ 9 -THC and by ≈ 25 % in cells exposed to 10 nM Δ 9 -THC. Lower doses of Δ 9 -THC (0.1 and 1 nM) or a shorter exposure time to the cannabinoid (6 h) were not effective. Down-regulation of δ opioid receptors was not observed in cells exposed for 24 h to pertussis toxin (PTX) and then treated for 24 h with 100 nM Δ 9 -THC. In cells that were exposed for 24 h to the cannabinoid, the ability of Δ 9 -THC and of the δ opioid receptor agonist [D-Ser 2 , Leu 5 , Thr 6 ]enkephalin to inhibit forskolin-stimulated cAMP accumulation was significantly attenuated. Prolonged exposure of NG 108-15 cells to 100 nM Δ 9 -THC produced a significant elevation of steady-state levels of δ opioid receptor mRNA. This effect was not observed in cells pretreated with PTX. The selective cannabinoid receptor antagonist SR 141716A blocked the effects elicited by Δ 9 -THC on δ opioid receptor desensitization, down-regulation and gene expression; thus indicating that these are mediated via activation of cannabinoid receptors. These data demonstrate the existence, in NG 108-15 cells, of a complex cross-talk between the cannabinoid and opioid receptors on prolonged exposure to Δ 9 -THC triggered by changes in signaling through G i and/or G 0 -coupled receptors.

Endothelin-1 response to mental stress in early ischemic lesions of the extremities due to systemic sclerosis

We studied circulating levels of endothelin-1, catecholamines and nitric oxide after a mental arithmetic test in 14 patients with early ischemic lesions of the extremities due to systemic sclerosis and slightly impaired peripheral vascular flow. The test induced an increase (P<0.01) in blood pressure, heart rate, endothelin-1 and catecholamine levels, whereas it did not change the low basal levels of nitric oxide. In healthy subjects (n=20) the test significantly (P<0.01) decreased endothelin-1 without affecting nitric oxide. The low basal levels of nitric oxide and the high plasma concentration of endothelin-1 after psychological stress cannot be explained by an impaired release from the limited ischemic lesions alone. This suggests a diffuse microvascular derangement that aggravates the course of peripheral microvascular ischemic lesions.

β-Endorphin modulation of pressor response to hyperventilation in hypertensive patients

After hyperventilation, systolic and diastolic blood pressure (BP) significantly decreased in 14 hypertensive patients (group 1), did not change in 9 (group 2) and increased in 8 (group 3). Basal BP, norepinephrine and dynorphin B levels were higher in group 1 than in groups 2 and 3. The decrease in BP after hyperventilation was associated with a decrease in plasma norepinephrine, Met-enkephalin and dynorphin B and an increase in beta-endorphin. Naloxone abolished the hyperventilation-induced BP and norepinephrine decreases. Our findings indicate that hyperventilation may select hypertensive patients with different sympatho-adrenergic activity and that the increase in beta-endorphin reduces BP response to hyperventilation in patients with high sympatho-adrenergic tone.

Synthesis and binding affinity of cis-(−)- and cis-(+)-N-ethyleneamino-N-nordeoxymetazocine and cis-(−)-N-normetazocine analogues at σ1, σ2 and κ opioid receptors

Abstract The synthesis of cis-(+)- and cis-(−)-N-ethyleneamino-N-nordeoxymetazocine and cis-(−)-N-normetazocine analogues is described and their affinities to σ1, σ2 and κ opioid receptors are evaluated. The cis-(+)-deoxy compounds displayed high σ/κ selectivity with nanomolar Ki values for σ1 receptors, whereas in the cis-(−)-N-normetazocine series the compound (−)-7b was found to bind with nanomolar affinity to the κ opioid receptor (Ki=21.5 nM). Compound (−)-7b showed good selectivity for the κ opioid receptor in comparison to the σ1 and σ2 sites and to the μ and δ opioid receptors. A correlation of the binding affinities between cis-(−)- and cis-(+)-N-deoxynormetazocine derivatives show that both isomers of the deoxy analogs have similar σ1 and σ2 binding profiles as the cis-(+)-N-normetazocine derivatives.

Plasma brain natriuretic peptide at rest and after adenosine-induced myocardial ischemia in normotensive and essential hypertensive patients with suspected coronary artery disease.

This study investigated plasma brain natriuretic peptide (BNP) levels in normotensive and hypertensive patients with suspected coronary artery disease during radionuclide pharmacological stress testing. Twenty-seven normotensive patients (15 males, aged 63.0+/-4.5 years and 12 females, aged 63.0+/-4.1 years) and 38 essential hypertensive patients (25 males, aged 63.3+/-3.3 years and 13 females, aged 64.6+/-2.6 years) with chest pain and exercise stress testing inconclusive for coronary artery disease underwent myocardial perfusion single-photon emission computed tomography (SPECT) using adenosine infusion. SPECT identified patients without (16 normotensive and 22 hypertensive) and patients with (11 normotensive and 16 hypertensive) transient perfusion defects. Basal BNP levels in normotensive patients without transient myocardial ischemia (3.1+/-1.2 fmol/ml) were significantly (P<0.01) lower than those observed in normotensive patients with transient ischemia (8.2+/-1.2 fmol/ml), whereas BNP levels in hypertensive patients without transient ischemia (8.2+/-1.0 fmol/ml) did not significantly differ from those in hypertensive patients with transient ischemia (8.1+/-2.0 fmol/ml). No significant difference was found in BNP levels between males or females either in normotensive or hypertensive patients without or with ischemia. Adenosine infusion did not significantly change BNP levels in any subject group without or with myocardial perfusion defects. Our findings show that increases in BNP allow early detection of myocardial ischemia in normotensive patients, but not in hypertensive patients with suspected coronary artery disease. Adenosine-induced myocardial ischemia does not affect BNP production already activated by coronary artery disease in normotensive patients and by hemodynamic changes in hypertensive patients.