Visualization of cyclic nucleotide binding sites in the vertebrate retina by fluorescence microscopy.

Cyclic nucleotides play a major role in cell signaling, especially in the nervous system. They act as cytoplasmic messengers in a wide range of physiological responses, but the spatial distribution of their sites of action within cells and tissues is not well-known. In the vertebrate retina, there is a class of well-characterized cGMP binding sites which control the permeability of cation channels in the rod outer segments (ROS), while cAMP is involved in several other systems in the inner retina. Biochemical studies of the cGMP-activated permeability in ROS have not distinguished between the subcellular compartments of disk and plasma membrane. By a new method using fluorescein-conjugated cyclic nucleotides, we have found strong cyclic GMP binding to the plasma membrane of the ROS, both on frozen sections of retina and in freshly isolated, leaky ROS. We also found a high density of cGMP binding sites on structures resembling the inner segment calycal processes. Little specific binding could be detected on the disk membranes or on any other retinal layer. In contrast, fluorescent cAMP did not label ROS, but gave a striking pattern of labeling on several deeper layers of the retina. These results suggest that the ROS plasma membrane has a much higher density of cGMP-controlled cation channels than the disk membranes, and point to other retinal layers where cAMP is likely to shape cellular responses. This method opens up novel morphological approaches to the study of cyclic nucleotide regulation.

MOKE setup exploiting a nematic liquid crystal modulator.

Here we report on the magneto-optical Kerr effect employing a nematic liquid crystal (LC) device as an optical modulator. This device allows performing intensity, phase, and polarization modulated measurements with a huge signal-to-noise ratio when compared to those obtained by means of an opto-mechanical chopper and a photo-elastic modulator. The results demonstrate that the optimal performance is achieved modulating the polarization state of the incident light by means of the LCs.

Extreme ultraviolet resonant inelastic X-ray scattering (RIXS) at a seeded free-electron laser.

In the past few years, we have been witnessing an increased interest for studying materials properties under non-equilibrium conditions. Several well established spectroscopies for experiments in the energy domain have been successfully adapted to the time domain with sub-picosecond time resolution. Here we show the realization of high resolution resonant inelastic X-ray scattering (RIXS) with a stable ultrashort X-ray source such as an externally seeded free electron laser (FEL). We have designed and constructed a RIXS experimental endstation that allowed us to successfully measure the d-d excitations in KCoF3 single crystals at the cobalt M2,3-edge at FERMI FEL (Elettra-Sincrotrone Trieste, Italy). The FEL-RIXS spectra show an excellent agreement with the ones obtained from the same samples at the MERIXS endstation of the MERLIN beamline at the Advanced Light Source storage ring (Berkeley, USA). We established experimental protocols for performing time resolved RIXS experiments at a FEL source to avoid X ray-induced sample damage, while retaining comparable acquisition time to the synchrotron based measurements. Finally, we measured and modelled the influence of the FEL mixed electromagnetic modes, also present in externally seeded FELs, and the beam transport with ~120 meV experimental resolution achieved in the presented RIXS setup.

Binding of two fluorescent cAMP analogues to type I and II regulatory subunits of cAMP-dependent protein kinases.

Binding of two long wavelength fluorescent cAMP analogues, 8-thioacetamido-fluorescein-cAMP (SAF-cAMP) and 8-thioacetamido-rhodamine-cAMP (SAR-cAMP), to the RI (from bovine muscle) and RII (from bovine heart) regulatory subunits of cAMP dependent kinases has been studied. Displacement of [3H]cAMP from RI and RII and equilibrium dialysis measurements show that the fluorescent nucleotides are high affinity ligands for the cAMP binding sites. The binding is characterized by complex fluorescence spectral and fluorescence anisotropy changes, more evident for the fluorescein than for the rhodamine derivative. The fluorescence excitation spectrum of the bound SAF-cAMP is characterized by the appearance of a red shifted shoulder at 500-510 nm excitation wavelength region. Any change of the bound/free ratio in a solution equilibrium is accompanied by changes in fluorescence and anisotropy signals which are best detected at suitable wavelengths. It is proposed that fluorescence and anisotropy changes can distinguish between binding to type B (slow dissociating) and A (fast dissociating) cAMP binding sites of regulatory subunits. Applications of the fluorescent nucleotides to kinase localization and cAMP determination in living cells are discussed.

Phosphodiesterase and GTPase in rod outer segments. Kinetics in vitro.

The hydrolysis of cyclic guanosine monophosphate (cyclic GMP) and of guanosine triphosphate (GTP) by the broken rods of the frog retina after a flash of light have been studied in vitro with a constant perfusion method. The activation has an onset apparently instantaneous as observed with the existing possible time resolution of 3 s. The activation is followed by a partial inactivation that does not bring the activity back to the pre-flash level. GTP or the non-hydrolysable guanyl-5'-ylimidodiphosphate (GMP-PNP) is required for the normal light-activation of the phosphodiesterase and in its absence both the speed of activation and the sensitivity are greatly reduced. The activation speed, the sensitivity (threshold at approx. 0.00004% bleaching), and the kinetic constants do not exclude a direct role in the process of excitation for the phosphodiesterase and suggest a subsidiary but as yet undefined role for the GTPase.

Light- and nucleotide-dependent increase in apparent viscosity in a suspension of retinal disks.

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.

H3-receptor antagonists: synthesis and structure-activity relationships of para- and meta-substituted 4(5)-phenyl-2-[[2-[4(5)-imidazolyl]ethyl]thio]imidazoles.

We report the synthesis, octanol/water partition coefficient (log P), dissociation constants (pKa), H3-receptor affinity (pKi in rat brain membranes, [3H]-N alpha-methylhistamine), and H3-antagonist potency (pA2 in guinea ileum, (R)-alpha-methylhistamine) of novel H3-receptor antagonists obtained by introducing a para or meta substituent on the phenyl ring of the lead compound 4(5)-phenyl-2-[[2-[4(5)-imidazolyl]ethyl]thio]imidazole (3a). The substituents were chosen to obtain broad and uncorrelated variation in their lipophilic, electronic, and steric properties. The log P values of the neutral species cover almost 3 orders of magnitude (from 1.40 to 4.11). The pKa,2 values (protonation of the 2-thioimidazole fragment) vary from 3.13 to 4.34, indicating that this fragment, which incorporates the so-called polar group common to many H3-receptor antagonists, is neutral at physiological pH. The compounds had pKi values in a range too narrow (from 7.28 to 8.03) to derive QSAR equations. In one case (3g), a biphasic displacement curve was observed (pKi,1 = 8.53; pKi,2 = 6.90). The pA2 values ranged 2 orders of magnitude (from 6.83 to 8.87) and yielded a QSAR model (PLS) indicating that antagonist potency depends parabolically on lipophilicity and is decreased by bulky para substituents. The compounds of this series, therefore, maintain a fair-to-good affinity for rat brain H3-receptor and a fair-to-good H3-antagonist potency on guinea pig ileum, although varying markedly in their lipophilicity. The series thus appears as a good candidate for pharmacokinetic optimization leading to brain-penetrating H3-receptor antagonists.

Search for a physiological role of cyclic GMP metabolism in the photoreceptors.

Data on the time-course of the light-activation of the cyclic GMP phosphodiesterase and of the GTPase, and results on the influence of cyclic GMP on the disc membrane permeability are presented. On the basis of the kinetic data, it is not possible to separate the light-activation of these two enzymes from the early steps of photoreceptor transduction. In addition, the cyclic GMP increases the permeability of the disc membranes, indicating that a decrease of the endogenous cyclic GMP concentration, consequent to the light-activation of the phosphodiesterase, can decrease the membrane permeability shortly after illumination.

cAMP-dependent protein kinase type RI is found in clusters in the rat detergent-insoluble neuronal fraction.

Different types of cAMP dependent regulatory subunits have been characterized in the mammalian brain: RI alpha and beta, RII alpha and beta. The subcellular distribution of RI subunits has been examined in the rat brain. Partial amino acid sequencing of tryptic fragments from the Triton insoluble pellet of the rat brain shows that cAMP dependent regulatory subunits type RI alpha are found in this fraction. Immunohistochemistry shows that Triton-insoluble RI subunits are concentrated to form clusters and this distribution is distinct from RII subunits. Immunohistochemistry and fluorescent cAMP labeling show that the clusters bind fluorescent cAMP analogues. These results suggest that the high local concentration of RI subunits can modulate cAMP distribution among different cellular compartments.

Visualization of detergent insoluble cyclic AMP-dependent protein kinase RIalpha aggregates in the rat brain.

Regulatory subunits of the cAMP dependent protein kinases are the most abundant receptor for cAMP in eukaryotic cells. Four isoforms of regulatory subunits (RIalpha and -beta, RIIalpha and -beta) have been distinguished. Distribution of the most abundant RII isoforms has been extensively studied in the brain, by immunohistochemistry and biochemical fractionation, while the least abundant RI isoforms have been neglected. In neurons most regulatory subunits are bound to the cytoskeleton. A protocol is presented that allows immunohistochemical and biochemical characterization of detergent-insoluble RI isoforms in the brain.

Light-induced infrared light scattering signal in the retina: effect of phosphodiesterase inhibitors.

Visible light changes IR light scatter through a toad retina. This signal presents three components: at low light intensity (100-400 bleached rhodopsins/rod) an early decrease in IR light scatter, of small amplitude, with time to peak of 1-6 s; at intermediate light intensity (1200-16,000 bleached rhodopsins/rod) a slow increase in IR light scatter, with time to peak of 10-30 s; at high light intensity (50,000-160,000 bleached rhodopsins/rod) a last increase in IR light scatter, with time to peak of 1 min. Light sensitivity, amplitude and time to peak of the last two components are increased by inhibitors (3-isobutyl-1-methyl-xanthine and papaverine) of the cyclic 3'5' guanosine monophosphate phosphodiesterase.

Pheromonally accelerated puberty is enhanced by previous experience of the same stimulus.

The influence of rearing conditions on pheromone-induced puberty acceleration was tested on Swiss mice. Litters were reared in one of three conditions: with either both parents, or with two females, or finally with two females in the presence of urinary pheromonal cues from adult males, which are known to induce puberty acceleration. Nine days after weaning the females were exposed to either prepubertal or adult male urine. In the groups reared with either both parents or with two females and the pheromonal cues from stranger males, females treated with adult male urine presented heavier uteri and more cornified vaginal smears than females reared in the same conditions but subsequently treated with prepubertal males urine. In the group reared simply with two females, the differences in both uterus weight and vaginal smears did not reach statistical significance. The data support the notion that early experience of pheromonal cues may influence the response to pheromones in a later period, even if the preweaning exposure to males had no direct influence on early signs of puberty onset.

Rat protein binding and cerebral phospholipid affinity of the H3-receptor antagonist thioperamide.

The binding of thioperamide, a known H3-receptor antagonist, to rat plasma and proteins and its affinity for rat cerebral phospholipids are investigated. Thioperamide is strongly bound to plasma proteins (95-80% at plasma concentrations of 3.5-400 micrograms mL-1), and its binding can be resolved into two components a high-affinity, saturable component and a non-specific component. The drug has a high affinity for cerebral phospholipids, with a partition coefficient of approximately 100 (log K = 2.06 +/- 0.14), which should promote brain penetration and accumulation. Protein binding and cerebral phospholipid affinity can suggest the explanation of some differences reported in the literature on thioperamide distribution data: at low plasma concentrations of the drug, its protein binding (95% at 3.5 micrograms mL-1) can prevent brain accumulation, while at higher concentrations the free plasma fraction suddenly increases (> 10% at 18 micrograms mL-1) and it allows passive distribution to lipophilic tissues such as brain tissue.

Synthesis, hypoglycemic and toxicological evaluation of new 1,2-benzisothiazolyl derivatives.

The synthesis of 1,2-benzisothiazol-3-ylguanidines, 1,2-benzisothiazol-3-ylbenzensulphonylureas and 1,2-benzisothiazol-3-ylbenzensulphonamides is described. Some of the new compounds showed moderate hypoglycemic activity but most of them caused serious acute toxic effects.

QSAR study on H3-receptor affinity of benzothiazole derivatives of thioperamide.

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.

HPLC detection of thioperamide from biological samples and its determination in rat blood and brain after systemic administration.

Thioperamide is a potent and selective antagonist on histamine H3 receptors. A method for its isolation and quantitation by HPLC from rat plasma and brain samples has been developed. Using this technique, thioperamide concentrations in rat plasma and brain were measured after systemic administration, in order to evaluate its persistence in blood and its ability to cross the blood-brain barrier. We observed that, at a dose of 60 mg/Kg, thioperamide undergoes a slow elimination from plasma, with a half-life of 10 hours, and can readily cross the blood-brain barrier.

Structure-property relationships on histamine H3-antagonists: binding of phenyl-substituted alkylthioimidazole derivatives to rat plasma proteins.

The binding of a series of H3-antagonists to rat plasma proteins was investigated by dialysis experiments, with RP-HPLC measurement of the free ligand. The series was composed of 4(5)-phenyl-2-[[2-[4(5)-imidazolyl]ethyl]thio]imidazoles having, on the phenyl ring, meta- and para-substituents, with different physico-chemical characteristics. As high protein binding had been proposed as being one of the features limiting brain access for the reference H3-antagonist thioperamide, the title series was employed to test the possibility of achieving lower protein binding by modulation of lipophilicity, while maintaining good receptor affinity. The compounds tested showed quotas of bound drug ranging from 60 to 97.5%, while for thioperamide a 78% bound drug quota was observed at high total concentrations, with a steep increase in bound percentage at lower concentrations. Two of the tested compounds, having a carboxamide substituent, showed lower protein binding compared to thioperamide over a wide range of total concentration, without a significant loss in affinity with respect to the parent compound. A strict dependence of protein binding on lipophilicity was observed, and a QSPR model was derived which could also account for the protein binding observed for thioperamide, while receptor affinity had been reported to be quite insensitive to phenyl ring substitution. It is therefore possible to modulate protein binding of these H3-antagonists, through lipophilicity adjustment, without losing receptor affinity; this finding could help in the design of new compounds with improved brain access.

Synthesis and binding assays of H3-receptor ligands.

The preparation of a representative group of derivatives of the known H3-antagonist thioperamide is described. Binding affinity for histamine H3-receptors of thioperamide and its derivatives, which were obtained by substitution on the imidazole ring, was measured on rat brain cortex synaptosomes. Competitive binding assays were performed with two different labelled ligands, the physiological agonist [3H]histamine ([3H]HA) and the potent H3-agonist N alpha-[3H]methyl-histamine ([3]NAMHA). We observed a remarkable difference in Ki values obtained versus the two labelled ligands, both for thioperamide and its derivatives. In particular, 5-methylthioperamide showed a considerable selectivity for the system recognized by [3H]NAMHA, being about 100 times more potent versus this system than versus the system recognized by [3H]HA. On the basis of these observations, we suggest that it is necessary to consider this difference in evaluating the affinity of new compounds for the H3-receptors.

Ligands of the histamine H3-receptor: new potent antagonists of the 2-thioimidazole type.

An overview of H3-receptor ligands is presented, with particular attention to antagonists. The protein binding of the classical H3-receptor antagonist thioperamide and its effect on in vivo distribution are discussed. A series of H3-receptor antagonists characterised by the presence of an imidazole ring, a spacer (ethylthio-, ethylamino-, propylthio- or propylamino-chain), a second heterocycle nucleus and a lipophilic group is described. Their H3-receptor antagonist potency has been measured on electrically stimulated guinea-pig intestine, and their affinity for central H3-receptor has been determined by competitive inhibition of [3H]N alpha-methylhistamine binding to rat cortex. Biphasic inhibition curves have been observed in some cases. Compounds endowed with interesting activity belong mostly to the class of 2-[[2-[4(5)-imidazolyl]ethyl]thio]imidazole, having a phenyl or a cyclohexyl group.

Plasma concentration and brain penetration of the H3-receptor antagonist thioperamide in rats.

Thioperamide is a potent and selective H3-receptor antagonist, whose in vivo effects have been reported after systemic administration. Some questions have arisen about its ability to cross the blood-brain barrier, since different experimental conditions have given different results in rats, namely a low brain/blood ratio at low doses (10 mg/Kg) and a much higher one at higher doses (60 mg/Kg). In this work we demonstrate the dose-dependence of thioperamide pharmacokinetics, measuring its plasma and cerebral levels after i.p. administration of different doses to rats. Both the plasma half-life and brain penetration of thioperamide resulted as being dose-dependent: when administered to 80 g body weight Wistar rats at 10 mg/Kg i.p., the drug has a short half life (120') and a rather poor brain penetration, but increasing the dose (to 20, 40 and 60 mg/Kg) gives rise to a prolongation of its persistence in the blood (up to 600' at highest dose) and a higher brain penetration. Also, the profile of the plasma concentration curve varies from the dose of 10 to that of 20 mg/Kg, passing from a mono-exponential decrease to a more complex one characterized by an apparent distribution phase. The different distribution processes can be interpreted in the light of thioperamide protein binding and affinity for lipophilic tissues: protein binding can prevent brain penetration (but not distribution to other tissues) at lower doses, while at higher doses the free plasma fraction increases and it can allow passive distribution to lipophilic tissues such as brain tissues. A re-distribution from these tissues and plasma is probably responsible for the strong increase in half-life at high doses.