Lower population of dopaminergic (DA) neurons is known to increase susceptibility to Parkinson\'s disease (PD), and our earlier study showed a lower yield of DA neurons in Leucine-Rich Repeat Kinase Isoleucine 1371 Valine (LRRK2-I1371V) mutation-carrying PD patient-derived induced Pluripotent Stem Cells (iPSCs). Although the role of Sonic Hedgehog (SHH) in DA neurogenesis of floor plate cells (FPCs) is known, the effect of LRRK2 mutations on SHH responsiveness of FPCs impacting DA neuronal yield has not been studied. We investigated SHH responsiveness of FPCs derived from LRRK2-I1371V PD patient iPSCs with regard to the expression of SHH receptors Patched1 (Ptch1) and Smoothened (Smo), in conjunction with nuclear Gli1 (glioma-associated oncogene 1) expression, intracellular Ca2+ rise, and cytosolic cyclic adenosine monophosphate (cAMP) levels upon SHH induction. In addition, we examined the mechanistic link with LRRK2-I1371V gain-of-function by assessing membrane fluidity and Rab8A and Rab10 phosphorylation in SH-SY5Y cells and healthy control (HC) FPCs overexpressing LRRK2-I1371V as well as FPCs. Although total expression of Ptch1 and Smo was comparable, receptor expression on cell surface was significantly lower in LRRK2-I1371V FPCs than in HC FPCs, with distinctly lower nuclear expression of the downstream transcription factor Gli1. HC-FPCs transfected with LRRK2-I1371V exhibited a similarly reduced cell surface expression of Ptch1 and Smo. Intracellular Ca2+ response was significantly lower with corresponding elevated cAMP levels in LRRK2-I1371V FPCs compared with HC FPCs upon SHH stimulation. The LRRK2-I1371V mutant FPCs and LRRK2-I1371V-transfected SH-SY5Y and HC FPCs too exhibited higher autophosphorylation of phospho LRRK2 (pLRRK2) serine1292 and serine935, as well as substrate phosphorylation of Rab8A and Rab10. Concurrent increase in membrane fluidity, accompanied by a decrease in membrane cholesterol, and lower expression of lipid raft marker caveolin 1 were also observed in them. These findings suggest that impaired SHH responsiveness of LRRK2-I1371V PD FPCs indeed leads to lower yield of DA neurons during ontogeny. Reduced cell surface expression of SHH receptors is influenced by alteration in membrane fluidity owing to the increased substrate phosphorylation of Rab8A and reduced membrane protein trafficking due to pRab10, both results of the LRRK2-I1371V mutation.

UNASSIGNED: Insect biogenic amines play important roles in mediating behavioral and physiological processes. They exert their effects by binding to biogenic amine receptors (BARs), which are specific receptor proteins in the G-protein-coupled receptor superfamily. BAR genes have been cloned and characterized from multiple model insects, including Drosophila melanogaster, Anopheles gambiae, Bombyx mori, Apis mellifera and Tribolium castaneum. However, relatively little work has addressed the molecular properties, expression profiles, and pharmacological characterization of BARs from other insects, including important pests.
RESULTS: In this study, we cloned 17 genes encoding putative biogenic amine receptor proteins from Plutella xylostella, a global pest of Brassica crops. These PxBAR genes were five octopamine receptors (PxOA1, PxOA2B1, PxOA2B2, PxOA2B3, and PxOA3), three tyramine receptors (PxTAR1A, PxTAR1B, and PxTAR2), four dopamine receptors (PxDOP1, PxDOP2, PxDOP3, and PxDopEcR), and five serotonin receptors (Px5-HT1A , Px5-HT1B , Px5-HT2A , Px5-HT2B , and Px5-HT7 ). All PxBARs showed considerable sequence identity with orthologous BARs, and phylogenetic analysis clustered the receptors within their respective groups while preserving organismal evolutionary relationships. We investigated their molecular properties and expression profiles, and pharmacologically characterized the dopamine receptor, PxDOP2.
CONCLUSIONS: Our study provides important information and resources on biogenic amine receptors from P. xylostella, which suggests potential target sites for controlling this pest species. © 2021 Society of Chemical Industry.

OBJECTIVE: The ability of the plant flavonol quercetin and its conjugated form quercetin-3-glucoside, compared to that of the anthocyanin cyanidin-3-glucoside, to interfere with 3\',5\'-cyclic adenosine monophosphate (cAMP) efflux was investigated in cultured human retinal pigment epithelial (HRPE) cells.
METHODS: HRPE cells were stimulated for a time course with 1 μM adrenaline, in the presence and absence of increasing concentrations of anthocyanins or flavonols, then intracellular and extracellular cAMP levels obtained from whole cells and cAMP synthetized by the activity of adenylate cyclase in cell membrane fractions were determined by radiochemical assay.
RESULTS: The treatment with either compound caused a significant lowering in extracellular cAMP concentrations deriving from a time course cell stimulation with 1 μM epinephrine. As to quercetin, the effect was shown to rely on the inhibition of cAMP efflux transporters. In the case of the glycoside, it was found to depend on the contrary on a reduction in the extent of epinephrine stimulation. Consistently, quercetin-3-glucoside inhibited the epinephrine-stimulated activity of adenylyl cyclase in membrane preparations, while quercetin was ineffective. The anthocyanin cyanidin-3-glucoside exerted similar effects as quercetin-3-glucoside.
CONCLUSIONS: Results strengthen the diverse effect of the glucosides versus the corresponding aglycones. Since differently from flavonols, anthocyanins are present in human plasma in their glycosylated form, the aglycone or glycoside forms of these plant secondary metabolites might therefore be utilized as synergistic regulators of cAMP homeostasis for therapeutical purposes.

The important regulator of cardiac function, cAMP, is hydrolyzed by different cyclic nucleotide phosphodiesterases (PDEs), whose expression and activity are not uniform throughout the heart. Of these enzymes, PDE2 shapes β1 adrenoceptor-dependent cardiac cAMP signaling, both in the right and left ventricular myocardium, but its role in regulating β2 adrenoceptor-mediated responses is less well known. Our aim was to investigate possible differences in PDE2 transcription and activity between right (RV) and left (LV) rat ventricular myocardium, as well as its role in regulating β2 adrenoceptor effects. The free walls of the RV and the LV were obtained from Sprague-Dawley rat hearts. Relative mRNA for PDE2 (quantified by qPCR) and PDE2 activity (evaluated by a colorimetric procedure and using the PDE2 inhibitor EHNA) were determined in RV and LV. Also, β2 adrenoceptor-mediated effects (β2-adrenoceptor agonist salbutamol + β1 adrenoceptor antagonist CGP-20712A) on contractility and cAMP concentrations, in the absence or presence of EHNA, were studied in the RV and LV. PDE2 transcript levels were less abundant in RV than in LV and the contribution of PDE2 to the total PDE activity was around 25% lower in the microsomal fraction of the RV compared with the LV. β2 adrenoceptor activation increased inotropy and cAMP levels in the LV when measured in the presence of EHNA, but no such effects were observed in the RV, either in the presence or absence of EHNA. These results indicate interventricular differences in PDE2 transcript and activity levels, which may distinctly regulate β2 adrenoceptor-mediated contractility and cAMP concentrations in the RV and in the LV of the rat heart.