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Despite cellular complexity, a limited number of small molecules act as intracellular second messengers. Protein kinase A (PKA) is the main transducer of the information carried by cyclic adenosine monophosphate (cAMP). Recently, cellular imaging has achieved major technical advancements, although the search for more specific and sensitive low-molecular-weight probes to explore subcellular events involving second messengers is still in progress. The convergent synthesis of a novel, fluorescent small molecule comprising the cAMP structure and a rhodamine-based fluorescent residue, connected through a flexible linker, is described here. The interaction motif of this compound with PKA was investigated in silico using a blind docking approach, comparing its theoretical binding energy with the one calculated for cAMP. Moreover, the predicted pharmacokinetic properties were also computed and discussed. The new probe was tested on three areas of the mouse central nervous system (parietal cerebral cortex, hippocampus, and cerebellar cortex) with different fixation methods demonstrating remarkable selectivity towards the PKA RIα subunit. The probe showed overall better performances when compared to other commercially available fluorescent cAMP analogues, acting at lower concentrations, and providing stable labeling.

The solid solution series KxBa1-xO2-δ (x < 0.41) is synthesized from stoichiometric amounts of Ba(NO3)2 and KO2 in liquid NH3 (-40 to -60 °C, 30 min).

Starting from the structure of thioperamide, a known H3-antagonist, a new series of compounds with a benzothiazole nucleus instead of the cyclohexylcarbothioamide moiety was synthesized. Various substituents, selected by experimental design, were introduced in position 6 of the benzothiazole nucleus, in order to change its physico-chemical characteristics. The lipophilicity of the synthesized compounds was measured by means of RP-HPLC, and their H3-receptor affinity was evaluated by competitive binding assays on rat cortex synaptosomes, with the labelled ligand N alpha-[3H]methylhistamine. A QSAR analysis was performed on the experimental data, using also substituent constants taken from the literature. The newly synthesized compounds showed lower H3-affinities than thioperamide; quantitative structure-activity relationships, described by models obtained with PLS and MRA techniques, were observed among benzothiazole derivatives. According to these relationships, any attempt to improve the potency of these compounds should involve the substitution of the benzothiazole moiety with less bulky and/or more flexible structures, which should also be less lipophilic and allow better electronic interactions with the binding site. 1-(Benzothiazol-2-yl)-4-[(1H)-imidazol-4-yl]piperidine represents a limit structure for H3-activity, since it seems impossible to improve its affinity by means of substitution in the studied position of the benzothiazole nucleus, as shown by predictions performed by a PLS model.

Guanosine 3/,5′-monophosphate (3′,5'GMP) activates an influx of calcium ions into vesicles of rod outer segments washed free of water-soluble proteins and filled with a hihg-affinity ligand for calcium (EDTA, Arsenazo III, aequorin). Addition of 3′,5′GMP activates calcium inflix without any appreciable delay, studied with methods that resolve 40 ms. A direct interaction between the cyclic nucleotide and an intrinsic membrane component is considered.

The apparently cooperative binding of 8-(5-thioacetamidofluorescein)-cGMP (SAF-cGMP) to cGMP-binding sites of the rod outer segments is regulated by Ca2+ in the 0.1-1 microM activity range. High Ca2+ reduces, and low Ca2+ increases the affinity of SAF-cGMP binding. This regulation involves only intrinsic membrane components. It is proposed that an allosteric regulation of cGMP binding by Ca2+ can contribute to photoreceptor potential adaptation.

Light triggers the cyclic nucleotide cascade in photoreceptor disk membranes. We report here that light-induced changes in the apparent viscosity of disk membrane suspensions can also be observed using either native disk membranes or washed membranes reconstituted with G protein and PDE. The viscosity changes are light- and GTP-dependent and require the presence of G protein and PDE. The magnitude of the viscosity change increases with increasing membrane concentration. Under the same conditions in which light elicits a change in viscosity, we observe a large increase in light scattering by the disk membrane suspension.