Stimulation of dopamine synthesis and activation of tyrosine hydroxylase by phorbol diesters in rat striatum

In rat striatal synaptosomes, 4 beta-phorbol 12-myristate 13-acetate (PMA) and 4 beta-phorbol 12,13-dibutyrate (PDBu), two activators of Ca2+-phospholipid-dependent protein kinase (protein kinase C) increased dopamine (DA) synthesis measured by following the release of 14CO2 from L-[1-14C] tyrosine. Maximal stimulation (21-28% increase of basal rate) was produced by 0.5 microM PMA and 1 microM PDBu. 4 beta-Phorbol and 4 beta-phorbol 13-acetate, which are not activators of protein kinase C, were ineffective at 1 microM. PMA did not change the release of 14CO2 from L-[1-14C]DOPA. Addition of 1 mM EGTA to a Ca2+-free incubation medium failed to affect PMA stimulation. KC1 (60 mM) enhanced DA synthesis by 25%. Exposure of synaptosomes to either PMA or PDBu prior to KC1 addition resulted in a more than additive increase (80-100%) of DA synthesis. A similar synergistic effect was observed when the phorbol diesters were combined with either veratridine or d-amphetamine but not with forskolin and dibutyryl cyclic AMP. Pretreatment of striatal synaptosomes with phorbol diesters produced an activation on of tyrosine hydroxylase (TH) associated with a 60% increase of the Vmax and a decrease of the Km for the pterine cofactor 6-methyl-5,6,7,8-tetrahydropterin. These results indicate that protein kinase C participates in the regulation of striatal TH in situ and that its activation may act synergistically with DA releasing agents in stimulating DA synthesis.     

Pertussis Toxin Attenuates D2 Inhibition and Enhances D1 Stimulation of Adenylate Cyclase by Dopamine in Rat Striatum

The response of adenylate cyclase to GTP and to dopamine (DA) was investigated in synaptic plasma membranes isolated from rat striatum injected with pertussis toxin, which inactivates the inhibitory guanine nucleotide-binding regulatory protein (Ni) of adenylate cyclase. Pertussis toxin treatment reverted the inhibitory effects on the enzyme activity elicited by micromolar concentrations of GTP and reduced by 50% the DA inhibition of cyclase activity via D2 receptors. The toxin treatment enhanced the net stimulation of enzyme activity by DA in the presence of micromolar concentrations of GTP. However, the stimulatory effect of the selective D1 receptor agonist SKF 38393 was not significantly affected. The data indicate that Ni mediates D2 inhibition of striatal adenylate cyclase and participates in the modulation of D1 stimulation of the enzyme activity by DA.     

Localization of growth arrest-specific genes on mouse Chromosomes 1, 7, 8, 11, 13, and 16

Growth arrest in NIH3T3 cells is associated with increased expression of a variety of mRNAs, several of which have been isolated as cDNA clones. Six of these growth arrest-specific (Gas) genes were mapped by following the inheritance of DNA restriction fragment length variants (RFLVs) associated with them in panels of recombinant inbred (RI) strains of mice and in the progeny of backcrosses both between laboratory mouse strains and between a laboratory strain and Mus spretus. The six genes are unlinked. Gas-1 maps to Chromosome (Chr) 13, Gas-2 to Chr 7, Gas-3 to Chr 11, Gas-4 to Chr 16, Gas-6 to Chr 8, and Gas-10 to Chr 1.     

Muscarinic Receptors Modulate Dopamine-Activated Adenylate Cyclase of Rat Striatum

We investigated the effect of acetylcholine (ACh) on the activation of adenylate cyclase by dopamine (DA) in a lysed synaptosomal preparation from rat striatum. ACh reduced both basal and the DA-activated adenylate cyclase with an apparent IC50 of approximately 1 microM. From a kinetic analysis it appeared that ACh reduced the Vmax for activation by DA but not the activation constant for DA. For most preparations the Vmax was reduced by 30-40%. The presence of atropine did not affect the activation of the enzyme by DA but it blocked the inhibition by ACh. Following 6-hydroxydopamine lesion of the nigrostriatal pathway, the enzyme became supersensitive to activation by DA and also more sensitive to inhibition by ACh. Inhibition of adenylate cyclase by ACh appeared to be rather specific for activation by DA, as ACh had no effect on activation of adenylate cyclase by the adenosine analogue N6-(L-2-phenylisopropyl)adenosine. These results indicate that some striatal muscarinic and dopaminergic receptors are probably coupled to the same adenylate cyclase domain. Moreover, they suggest a biochemical model for the dynamic balance of cholinergic and dopaminergic neurons that innervate the striatum.     

Activation of adenosine A1 receptor by N6-(R)-phenylisopropyladenosine (R-PIA) inhibits forskolin-stimulated tyrosine hydroxylase activity in rat striatal synaptosomes

We investigated the effect of the relatively selective A1 adenosine receptor agonist N6-(R)-phenylisopropyladenosine (R-PIA) on tyrosine hydroxylase activity (TH) of synaptosomes obtained from rat striatum. TH activity was assayed in supernatant obtained following sonication and centrifugation of the tissue preincubated with the test compounds. R-PIA produced a modest decrease of basal enzyme activity, but significantly reduced the activation of the enzyme by submaximal (0.1-0.5 microM) concentrations of forskolin (FSK) a stimulator of adenylate cyclase. The IC 50 value of R-PIA was 17 nM and the maximal inhibition corresponded to 30-40% decrease of the enzyme activity stimulated by FSK. The S-isomer of PIA failed to affect TH activity under control and stimulated conditions. Moreover, the inhibitory effect of R-PIA was completely antagonized by 8-cyclopentyl- 1,3 -dimethylxanthine, an adenosine receptor blocker. R-PIA inhibited both basal and FSK-stimulated adenylate cyclase activity. These results indicate that in striatal dopaminergic terminals TH activity can be modulated in an inhibitory manner by activation of presynaptic A1 adenosine receptors.     

Effects of Sirolimus treatment on patients with β-Thalassemia: Lymphocyte immunophenotype and biological activity of memory CD4+ and CD8+ T cells

Inhibitors of the mammalian target of rapamycin (mTOR) have been proposed to improve vaccine responses, especially in the elderly. Accordingly, testing mTOR inhibitors (such as Sirolimus) and other geroprotective drugs might be considered a key strategy to improve overall health resilience of aged populations. In this respect, Sirolimus (also known as rapamycin) is of great interest, in consideration of the fact that it is extensively used in routine therapy and in clinical studies for the treatment of several diseases. Recently, Sirolimus has been considered in laboratory and clinical studies aimed to find novel protocols for the therapy of hemoglobinopathies (e.g. β-Thalassemia). The objective of the present study was to analyse the activity of CD4⁺ and CD8⁺ T cells in β-Thalassemia patients treated with Sirolimus, taking advantages from the availability of cellular samples of the NCT03877809 clinical trial. The approach was to verify IFN-γ releases following stimulation of peripheral blood mononuclear cells (PBMCs) to stimulatory CEF and CEFTA peptide pools, stimulatory for CD4⁺ and CD8⁺ T cells, respectively. The main results of the present study are that treatment of β-Thalassemia patients with Sirolimus has a positive impact on the biological activity and number of memory CD4⁺ and CD8⁺ T cells releasing IFN-γ following stimulation with antigenic stimuli present in immunological memory. These data are to our knowledge novel and in our opinion of interest, in consideration of the fact that β-Thalassemia patients are considered prone to immune deficiency.     

Bimodal regulation of cyclic AMP by muscarinic receptors. Involvement of multiple G proteins and different forms of adenylyl cyclase

In membranes of rat olfactory bulb, muscarinic receptor agonists stimulate basal adenylyl cyclase activity . This response is inhibited by a number of muscarinic receptor antagonists with a rank order of potency suggesting the involvement of the M4 muscarinic receptor subtype. The stimulatory effect does not require Ca2+ and occurs independently of activation of phosphoinositide hydrolysis. Pretreatment with pertussis toxin completely prevents the muscarinic stimulation of adenylyl cyclase, indicating the participation of G proteins of the Gi/Go family. Immunological impairment of the G protein, Gs, also reduces the muscarinic response, whereas concomitant activation of Gs-coupled receptors by CRH or VIP results in a synergistic stimulation of adenylyl cyclase activity. Although these data suggest a role for Gs, a body of evidence indicates that the muscarinic receptors do not interact directly with this G protein. Moreover, the Ca2+/calmodulin (Ca2+/CaM)- and forskolin-stimulated enzyme activities are inhibited by muscarinic receptor activation in a pertussis toxin-sensitive manner and with a pharmacological profile similar to that observed for the stimulatory response. These data indicate that in rat olfactory bulb M4 muscarinic receptors exert a bimodal control on cyclic AMP formation through a sequence of events that may involve activation of Gi/Go proteins, synergistic interaction with Gs and differential modulation of Ca2+/CaM-independent and -dependent forms of adenylyl cyclase.     

Properties of Muscarinic-Stimulated Adenylate Cyclase Activity in Rat Olfactory Bulb

The muscarinic stimulation of adenylate cyclase activity in rat olfactory bulb was characterized, with the aim of elucidating the nature of the molecular mechanism involved. Carbachol (CCh) stimulated the enzyme activity in either crude or purified cell membrane preparations and increased cyclic AMP accumulation in miniprisms of olfactory bulb. The CCh stimulation of adenylate cyclase activity displayed a fast onset and was rapidly reversed by addition of atropine. The stimulation was associated with an increase in the apparent Vmax of the enzyme, with no change in the Km for Mg-ATP. The affinity of the enzyme for Mg2+ was enhanced by CCh. The muscarinic effect required GTP at concentrations higher than those needed for enzyme stimulation with either l-isoproterenol or vasoactive intestinal peptide. Moreover, contrary to the beta-adrenergic stimulation, the muscarinic effect disappeared when guanosine 5'-O-(3'-thiotriphosphate) was substituted for GTP. In vivo treatment of olfactory bulbs with pertussis toxin completely prevented the muscarinic stimulation of adenylate cyclase, whereas cholera toxin was without effect. These results indicate that in rat olfactory bulb muscarinic receptors increase adenylate cyclase activity by interacting with a pertussis toxin-sensitive GTP-binding protein different from the stimulatory GTP-binding protein.     

Body site‐specific genetic effects influence naevus count distribution in women

Body site is highly relevant for melanoma: it affects prognosis and varies according to the patient's sex. The distribution of naevi, a major risk factor for melanoma, at different body sites also varies according to sex in childhood. Using naevus counts at different body sites in 492 unrelated adults from both sexes, we observed that women have an increased number of naevi on the lower limbs compared to men (p = 8.5 × 10^?5), showing that a high naevus count on this site persists from childhood throughout life. Then, using data from 3,232 twins, we observed, in women, the lowest naevus count heritability on the trunk (26%), and the highest on the lower limbs (69%). Finally, we showed that, in 2,864 women, six genomic loci previously associated with both naevus count and melanoma risk (IRF4, DOCK8, MTAP, 9q31.2, KITLG and PLA2G6) have an effect on naevus count that is body site‐specific, but whose effect sizes are predominantly stronger on the lower limbs. Sex‐specific genetic influence on naevus count at different sites may explain differences in site‐specific melanoma incidence as well as prognosis between sexes.