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Protein kinase A (PKA) are tetramers of two catalytic and two regulatory subunits, docked at precise intracellular sites to provide localized phosphorylating activity, triggered by cAMP binding to regulatory subunits and subsequent dissociation of catalytic subunits. It is unclear whether in the brain PKA dissociated subunits may also be found. PKA catalytic subunit was examined in various mouse brain areas using immunofluorescence, equilibrium binding and western blot, to reveal its location in comparison to regulatory subunits type RI and RII. In the cerebral cortex, catalytic subunits colocalized with clusters of RI, yet not all RI clusters were bound to catalytic subunits. In stria terminalis, catalytic subunits were in proximity to RI but separated from them. Catalytic subunits clusters were also present in the corpus striatum, where RII clusters were detected, whereas RI clusters were absent. Upon cAMP addition, the distribution of regulatory subunits did not change, while catalytic subunits were completely released from regulatory subunits. Unpredictably, catalytic subunits were not solubilized; instead, they re-targeted to other binding sites within the tissue, suggesting local macromolecular reorganization. Hence, the interactions between catalytic and regulatory subunits of protein kinase A consistently vary in different brain areas, supporting the idea of multiple interaction patterns.

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 in vitro and ex vivo antiplatelet effects of 2-amino-1,2-benzisothiazolin-3-one (1) are compared with those of its parent compound 1,2-benzisothiazolin-3-one (2) and with acetylsalicylic acid (ASA) against different agonists. 2-Amino-1,2-benzisothiazolin-3-one inhibits adenosine diphosphate (ADP)-, arachidonic acid (AA)- and collagen-induced human platelet aggregation in vitro, with IC50 values of 8.90 × 10 -5 , 1.50 × 10 -6 and 5.11 × 10 -8 mol/l, respectively. The strong inhibitory activity is significant not only for collagen but also for AA-induced aggregation. The same compound inhibits ex vivo collagenand particularly AA-induced rabbit platelet aggregation at the tested dose of 10 mg/kg i.m. In view of the potential use of 2-amino-1,2-benzisothiazolin-3-one as antithrombotic agent, the log P values for both 1,2- benzisothiazolin-3-one derivatives 1 and 2 are determined, to gain an understanding of the significance of the 2-amino group in the 1,2-benzisothiazolin-3-one moiety with respect to the biological activity under study.

Urine is one of the major media for intraspecific chemical communication in mice. The urination pattern is dependent both on the mice's hormonal and social status. The urination pattern and the morphology of the urinary tract were examined in mice following hormonal manipulations. In the first experiment, we compared pairs of intact and castrated males: intact males urinated earlier when exposed to a new environment, with a greater number of drops that were smaller than those of castrated males. In the second experiment, groups of intact males, castrated, testosterone-supplemented castrated, and isolated intact males were compared. The micturition pattern of isolated intact males consisted of numerous small droplets of urine, with a high volume of urine retained in the bladder after voiding. This also applied to grouped intact males and testosterone-treated castrated mice, while castrated mice voided a larger fraction of bladder content. Bladder weight was higher in intact males and testosterone-treated castrated males, as compared to castrated males. In the third experiment, ovary-intact and testosterone-treated intact females were compared. Testosterone-treated ovary-intact females retained a larger quantity of urine in the bladder and also had a larger bladder compared to ovary-intact females. Testosterone thus induces the morphological modifications of the urinary tract necessary for the dominant male urination pattern, which is an increase in postvoid urinary residual volume and bladder weight. As evidenced from the comparison of histological sections from intact, castrated, and testosterone-treated castrated males, the increase in bladder weight was mainly due to the bladder muscular mass.

ABSTRACT Glioblastoma is the most lethal brain tumor. The poor prognosis results from lack of defined tumor margins, critical location of the tumor mass and presence of chemo- and radio-resistant tumor stem cells. The current treatment for glioblastoma consists of neurosurgery, followed by radiotherapy and temozolomide chemotherapy. A better understanding of the role of molecular and genetic heterogeneity in glioblastoma pathogenesis allowed the design of novel targeted therapies. New targets include different key-role signaling molecules and specifically altered pathways. The new approaches include interference through small molecules or monoclonal antibodies and RNA-based strategies mediated by siRNA, antisense oligonucleotides and ribozymes. Most of these treatments are still being tested yet they stay as solid promises for a clinically relevant success.

The binding of thioperamide, a known H3-receptor antagonist, to rat plasma 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 μg 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 μg mL−1) can prevent brain accumulation, while at higher concentrations the free plasma fraction suddenly increases (> 10% at 18 μg mL−1) and it allows passive distribution to lipophilic tissues such as brain tissue.

The pirenzepine-related analogue, nuvenzepine, and the antagonists selective for the three muscarinic receptor subtypes 4-DAMP (M, and M3 receptors), pirenzepine (M 1 receptors), methoctramine (M 2 receptors) have been tested on rabbit isolated small and large intestinal regions provided with spontaneous motor activity. The employed drugs differently affected intestinal motility patterns. The ileum pendular movements as well as the proximal colon and taenia coli tone, spike amplitude and frequency were concentration-dependently inhibited by the compounds here employed. Their pIC 50 values followed the rank order of potency generally reported for the involvement of the M 3 muscarinic receptors (4-DAMP≥atropine>nuvenzepine≥pirenzepine>methoctramine). Unlike nuvenzepine and the other antimuscarinics assayed, the M 1 selective antagonist pirenzepine, at nanomolar concentrations, was able to enhance the proximal taenia coli motility patterns suggesting that a M 1 -inhibitory pathway might operate in the physiological modulation of taenia coli motility. At variance with longitudinal ileum and colon contractile activity, proximal circular colon motility was resistant to muscarinic as well as to α 1 -, H 1 -, 5-HT-blockade indicating that NANC neuronal mechanisms could act at this level. In summary, these data provide evidence that, at intestinal level, nuvenzepine is almost completely devoid of reliable M 1 -linked effect thus possessing a different pharmacological selectivity at muscarinic receptor subtypes with respect to pirenzepine. Furthermore, it emerges that rabbit spontaneous small and large intestinal motility is probably driven by different physiological mechanisms regional-related. The peculiar circular colon refractoriness deserves further studies to be extended to the human tissue.

The intracellular second messenger adenosine 3',5'-cyclic monophosphate (cAMP) acts mainly through cAMP-dependent protein kinases (PKA). In mammals and reptiles, the PKA regulatory isoforms (RI and RII) are differentially distributed among the various brain areas and cell types, according to the age of the animal. Since PKA distribution may be an additional marker for homologous areas, PKA regulatory subunit types RI and RII were examined in the chicken brain, a species not yet investigated. Chicken brains were examined from prehatching to adult age, by means of immunohistochemistry and biochemical characterization. Most PKA regulatory subunits were segregated in discrete non-soluble clusters that contained either RI or RII. While RII aggregates were present also in non-neuronal cells, RI aggregates were detected only in neurons of some brain areas that are mainly related to the telencephalon. They appeared later than RII aggregates; their presence and location varied during development. RI aggregates were detected first in the olfactory bulb, around embryonic day 14; within 3 days they appeared in the hyperpallium and nidopallium, where the most intense labeling was observed in the perihatching period. Fainter RI aggregates persisted up to 3 years in the olfactory bulb and nidopallium caudale. Less intense RI aggregates were present for a shorter time, from 2 weeks to 3 months, in the septal nuclei, thalamic medial nuclei, periventricular hypothalamus, optic tectum periventricular area, brainstem reticular formation and spinal cord substantia gelatinosa. RI aggregates were not detected in many brain areas including the arcopallium, striatum and cranial nerve nuclei. RII distribution showed less variation during development. From embryonic day 12, some insoluble RII aggregates were detected in the brain; however, only minor modifications were observed in positive structures once they started to harbor insoluble RII aggregates. The present results suggest that the distribution of PKA aggregates may assist in characterizing phylogenetically homologous structures of the vertebrate central nervous system and may also unravel biochemical differences among areas considered homologous.

The effects of diltiazem, a drug which inhibits the calcium channels in cardiac muscle as well as the light-sensitive channels in photoreceptor cells, were studied on ionic fluxes in both membrane and intact cell preparations. Diltiazem nonselectively increased the ionic permeability to both anions and cations in photoreceptor rod outer segment and synaptic membrane vesicles as well as in intact erythrocytes. Under our conditions, the estimated threshold for the diltiazem effect varied between 12.5 and 200 microM. In each case the concentration dependence exhibited the sigmoidal shape characteristic of positive cooperativity. The effect of diltiazem on ionic fluxes from phospholipid vesicles were strongly influenced by phospholipid composition and membrane charge. By contrast, diltiazem inhibited the efflux of 86Rb from photoreceptor cells of intact aspartate-isolated retina, an effect opposite to that of diltiazem on ionic permeabilities in photoreceptor membrane vesicle preparations. These data raise serious doubts on the specificity of diltiazem as a calcium channel blocker or as a cGMP channel blocker when used at concentrations higher than 10 microM.

Differential diagnosis of brain tumor types is mainly based on cell morphology and could benefit from additional markers. The cAMP second-messenger system is involved in regulating cell proliferation and differentiation and is conceivably modulated during cancer transformation. The cAMP second-messenger system mainly activates protein kinases, which are in part docked to cytoskeleton, membranes, or organelles by anchoring proteins, forming protein aggregates that are detergent insoluble and not freely diffusible and that are characteristic for each cell type. The intracellular distribution of the detergent-insoluble regulatory subunits (R) of the cAMP-dependent protein kinase has been examined in mouse and rat glioma cells both in vitro and in vivo by immunohistochemistry. In normal rodent brains, the RIIalpha regulatory subunit is detergent insoluble only in ependymal cells, while in the rest of the brain it is present in soluble form. Immunohistochemistry shows that in both mouse and rat glioma cell lines, RIIalpha is mainly detergent insoluble. RIIalpha is localized close to the nucleus, associated with smooth vesicles in the trans-Golgi network area. Both paclitaxel and vinblastine cause a redistribution of RIIalpha within the cell. Under conditions that increased intracellular cAMP, apoptosis of glioma cells was observed, and it was accompanied by RIIalpha redistribution. Also in vivo, detergent-insoluble RIIalpha can be observed in mouse and rat gliomas, where it delineates the border between normal brain tissue and glioma. Therefore, intracellular distribution of detergent-insoluble RIIalpha can assist in detecting tumor cells within the brain, thus making the histologic diagnosis of brain tumors more accurate, and may represent an additional target for therapy.

In the presence of G protein and phosphodiesterase, GTP induces aggregation of phospholipid-free rhodopsin-detergent micelles or rhodopsin reconstituted in phospholipid vesicles. The net electrical charge of the vesicle is not critical to the aggregation process since this phenomenon is not altered by reconstitution with phospholipids with different charge. The aggregation process is observed by monitoring changes in the light-scattering properties of the detergent micelles or vesicle suspension and by phase-contrast microscopy. The lowest light intensity which triggers the aggregation process and concomitant light-scattering changes in a rhodopsin-detergent micellar suspension bleaches 6% rhodopsin. Under these conditions, the signal saturates at 30% rhodopsin bleaching. The aggregation process appears likely to depend on the protein-protein interaction, and the presence of a disk membrane is not necessary for this process.

cAMP is a universal second messenger. In eucaryotes it acts mainly via protein kinases composed of regulatory (R) and catalytic subunits; their subcellular distribution may differ according to the cell type. In rodent brain, peculiar detergent-insoluble RIalpha aggregates were previously described in neurons of areas related to the limbic system, while RIIbeta is more evenly distributed also in non-nervous cells. It is unclear whether the regional distribution of regulatory subunits is typical of mammalian brain. Western blots and immunohistochemistry showed that in lizard brains a large fraction of the cAMP-dependent protein kinase regulatory isoforms is insoluble, as in mammals. Insoluble RIalpha and RII regulatory isoforms were not evenly distributed but organized in clearly separated aggregates. Numerous RII aggregates were present in almost all brain regions and were found also in non-nervous cells. As shown by immunohistochemistry and equilibrium binding of fluorescently tagged cAMP, RIalpha aggregates were restricted to neurons of some brain regions: telencephalon, particularly medial cortical areas, dorsal ventricular ridge, olfactory pathways, medial hypothalamus and cerebellar granular layer were intensely labelled. A very weak RIalpha labelling was detected in the brainstem reticular formation, in the periaqueductal gray and in the spinal cord dorsal horn. A similar distribution of RIalpha aggregates was also found in turtle brains. Their distribution is reminiscent of that observed in mammals, although with some differences in relative intensity and persistence. The supramolecular organization of the RIalpha isoform may help in establishing homologies and differences between brain areas involved in visceroemotional control.

Chemical signals modulate aggressive behavior in mice. For example, male urinary cues enhance aggression against other adults: a resident mouse attacks a male but not a castrated intruder, unless it is anointed with male urine. Our purpose was to understand whether molecules excreted with urine also act as aggression triggers in a different context. Therefore, the effect of urine, or molecules purified from urine, voided by different animals (males or females), was tested on the aggression of male mice against pups. Latency to the first attack, percentage of pups receiving the first attack, and percentage of attacked pups after 5 and 15 min were recorded. At variance with intermale aggression, male urinary chemosignals sprayed on pups reduced infanticide, while female urine did not. Male urine also delayed infanticide when compared to female urine. Pups anointed with low molecular weight dialyzed urine and with the high molecular weight protein fraction were attacked later than controls. Pups anointed with Major Urinary Proteins (MUPs) also were attacked later. The volatiles retained by MUPs act in the same way as adult male urine. MUPs and their ligands did not modify biting of food items. The results show that mice do not perceive male chemosignals as compulsory aggression triggers but rather can consistently and differentially shape their behavior in response to the same molecules according to different contextual events.

Psychiatric or depressed patients show alterations in both olfactory projection areas and mucosa. In rodents, ablation of olfactory bulbs causes a depression-like syndrome, useful to test antidepressant agents. We studied in mice the behavioral symptoms and neuroanatomical correlates after mucosal damage or ablation of the olfactory bulb. Our results are based on a battery of tests exploiting anxious, aggressive, and depressive behavior, on morphological and immunohistochemical analysis. We found similar results in both sensory-damaged and bulbectomized animals, with a behavioral dissociation concerning different forms of aggression. These findings do not support a simple downregulation of social interactions in damaged mice. The most prominent modifications in the brains of sensory damaged and bulbectomized mice are detected in the subventricular zone (SVZ), the source area of neural stem cells, and in the content of cAMP-dependent protein kinase within the amygdala, suggesting a central role of this structure in the functional modulation of behavior.

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.

In eukaryothes, the second messenger cAMP regulates many cellular functions by binding to the regulatory subunits of cAMP-dependent protein kinases, and releasing the catalytic subunits. In the mammalian brain all four regulatory isoforms (RIalpha and beta, RIIalpha and beta) are present. Apparently, they are simple inhibitors of the catalytic subunits. It is still unclear why four isoforms are needed, but possibly they can target kinase activity at precise intracellular locations. Therefore, we examined the distribution of the insoluble regulatory isoforms in rat and mouse brains during prenatal (from embryonic day 8) and postnatal development up to senescence (13 months), via immunohistochemistry. RIIalpha labelling is always restricted to the ventricular ependyma. Punctated RIIbeta labelling is observed in the embryo from early stages of development, and is mainly localized in the cortical plate. After birth, punctate RIIbeta labelling is present throughout almost the whole brain, often observed in proximity of neurofilaments. It shows different characteristics and relationships to the other isoforms: for example in the CA1 hippocampal field, RIIbeta is substituted by RIalpha 2 weeks after birth, while in CA2 it persists for life. In other regions, as in the reticular formation, RIIbeta and RIalpha aggregates are found in the same cell, although clearly segregated. The different regulatory isoforms show distinct patterns of distribution that change consistently during development. A careful characterization of second messenger systems may be as useful as the study of neurotransmitters to understand neuronal properties and their modifications during development, so as to relate biochemical to functional properties.

Poor micturition control may cause profound distress, because proper voiding is mandatory for an active social life. Micturition results from the subtle interplay of central and peripheral components. It involves the coordination of autonomic and neuromuscular activity at the brainstem level, under the executive control of the prefrontal cortex. We tested the hypothesis that administration of molecules acting as reuptake inhibitors of serotonin, noradrenaline or both may exert a strong effect on the control of urine release, in a mouse model of overactive bladder. Mice were injected with cyclophosphamide (40 mg/kg), to increase micturition acts. Mice were then given one of four molecules: the serotonin reuptake inhibitor imipramine, its metabolite desipramine that acts on noradrenaline reuptake, the serotonin and noradrenaline reuptake inhibitor duloxetine or its active metabolite 4-hydroxy-duloxetine. Cyclophosphamide increased urine release without inducing overt toxicity or inflammation, except for increase in urothelium thickness. All the antidepressants were able to decrease the cyclophosphamide effects, as apparent from longer latency to the first micturition act, decreased number of urine spots and volume of released urine. These results suggest that serotonin and noradrenaline reuptake inhibitors exert a strong and effective modulatory effect on the control of urine release and prompt to additional studies on their central effects on brain areas involved in the social and behavioral control of micturition.

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.

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.

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.

The attractive properties of male urinary pheromones were tested on adult or prepubertal male and female mice. An androgen-dependent protein is present in adult male urine (major urinary protein, MUP) which has been suggested to be a pheromone-binding protein. We tested the pheromonal properties of the protein-bound volatiles in a test of attractiveness. These molecules, that co-purify with MUP, attract females and repel adult males. In prepubertal animals, females are repelled and males are attracted by the same stimuli. These results are similar to those obtained by others with adult male whole urine. Therefore MUP binds molecules with a pheromonal activity, and these molecules are sufficient to act as male signals.

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.

The recent availability of potent and selective ligands, namely R-(alpha)-methylhistamine and thioperamide, led to conclusive progresses as regards histamine H3-receptor knowledge. The aim of this work is to investigate by in vitro tests the pharmacological properties of new amino and methyl derivatives of the H3-antagonist thioperamide. Such original compounds, developed by the modulation of the thioperamide imidazolyl moiety, were assayed at guinea-pig ileal contractile H1-, atrial chronotropic H2- and enteric neuronal H3-receptors. None of the drugs exhibited interaction with H1 or H2 sites. On electrically stimulated ileum, two of the thioperamide methyl derivatives competitively antagonized the inhibitory effect of the H3-agonist R-(alpha)-methylhistamine. On the basis of the Schild analysis, the more active isomer (compound IV) displayed an affinity at H3-receptors only five times lower than thioperamide. These results could contribute to elucidate further the structural features required to develop potent and selective H3-antagonists. On the other hand, to prove the hypothesized apparent heterogeneity between peripheral and central H3-sites, as emerged by pharmacological and binding studies, autoradiographic investigations are in progress.

Visible light activates a large guanosine cyclic 3',5'-phosphate (cGMP)- and phosphodiesterase (PDE)-dependent infrared light-scattering change in suspensions of photoreceptor disk membranes. Reconstitution experiments show that this signal requires bleached rhodopsin, G protein (three polypeptide subunits of Mr 39 000, 37 000, and 6000 which comprise the GTPase), phosphodiesterase, cGMP, and GTP. The lowest light intensity which elicits the light-scattering signal bleaches 0.002% rhodopsin. cGMP and GTP hydrolysis occurs more slowly than the initial phase of the scattering signal, and the kinetics of nucleotide hydrolysis do not correlate with any phase of the signal. Hydrolysis-resistant analogues of cGMP and GTP support the initial decreasing phase of the signal. Thus, the signal apparently depends upon nucleotide binding rather than hydrolysis. Microscopic observations made under the same conditions as light-scattering experiments show that vesicle-vesicle aggregation and disaggregation occur. The data suggest that light and nucleotide activations of the cyclic nucleotide cascade enzymes are responsible for the vesicle aggregation process and nucleotide hydrolysis for vesicle disaggregation. The vesicle aggregation-disaggregation phenomenon appears likely to be the physical basis of the cGMP- and PDE-dependent changes in infrared transmission.

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.

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.

1. The diffusion of sodium, potassium and rubidium (not chloride) ions across the disk membrane is increased by cyclic guanosine monophosphate (cyclic GMP). 2. The increase is greater for sodium than for rubidium in the 0.01-0.1 mM concentration range. 3. Cyclic adenosine monophosphate (cyclic AMP) is less efficient than cyclic GMP; GMP and guanosine triphosphate are without effect. 4. The effect is present with either 1.8 mM calcium ions or 4 mM-EGTA in the perfusion fluid. 5. The presence of the cyclic GMP phosphodiesterase on the disk membranes is not needed for this effect. 6. The effect is present in both unbleached and fully bleached membranes.

Electrical photoresponses of rods in the isolated toad retina were recorded during ionic manipulations of the Na+-free extracellular medium. In the presence of a concentration of external Ca2+ above 10(-5) M, voltage photoresponses were observed only in the presence of external Na+ or Li+. When external Ca2+ was reduced below 10(-6) M, voltage photoresponses of normal polarity could be detected even in the absence of Na+ or Li+, but in the presence of external Mg2+. In the presence of normal extracellular Ca2+ hyperpolarizing photoresponses were observed even in the absence of Na+ or Li+, provided small amounts of phosphodiesterase inhibitors (IBMX, RO 20-1724, papaverine, caffeine, theophylline) were added to the perfusate. Responses obtained in low-Na+ IBMX solutions required the presence of millimolar amounts of a variety of divalent cations, among which Mn2+ and Ba2+ were the most effective. When the concentration of both external Ca2+ and Mg2+ was reduced to micromolar amounts, depolarizing photoresponses were observed. In these conditions measurements with radioactive tracers showed a light-modulated efflux of 42K+ or 86Rb+. The light-modulated 42K+ or 86Rb+ efflux was halved by 2 X 6 mM-external K+ and was completely blocked when K+ was raised above 10 mM. These results show that ionic movements through light-sensitive channels are controlled by Ca2+ and Mg2+ and possibly also be the intracellular level of cyclic nucleotides. Moreover, the movement of ions through the light-sensitive channel, does not obey the independence principle.

Under conditions in which large guanosine cyclic 3',5'-phosphate (cGMP)- and phosphodiesterase (PDE)-dependent changes in near-infrared transmission and vesicle aggregation and disaggregation occur, we have observed a striking change in the binding of PDE to rod disk membranes. The change in PDE binding is nucleotide and light dependent as are the light-scattering changes. The cGMP- and PDE-dependent light-scattering signal can be produced by a 500-nm light flash which bleaches 1/(1 X 10(7] rhodopsin molecules. Mg ions are an essential cofactor for the nucleotide-dependent PDE binding and light-scattering changes. 3-Isobutyl-1-methylxanthine and other competitive inhibitors of PDE hydrolytic activity support increased PDE binding to the disk membrane, vesicle aggregation, and the light-scattering signal. However, treatments which block GTP-dependent activation of PDE hydrolytic activity (colchicine, GDP, or ethylenediaminetetraacetic acid) also block these phenomena. Thus, GTP-dependent activation of PDE rather than its hydrolytic activity appears to be correlated with the light-scattering signal.

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 levl. GTP or the nonhdrolysable guanyl-5′-ylimidodiphosphate (GMP- P N P ) 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.

Background Olfaction is often affected in parkinsonian patients, but dopaminergic cells in the olfactory bulb are not affected by some Parkinson-inducing drugs. We investigated whether the drug MPTP produces the olfactory deficits typical of Parkinson and affects the olfactory bulb in mice. Findings Lesioned and control mice were tested for olfactory search, for motor and exploratory behavior. Brains and olfactory mucosa were investigated via immunohistochemistry for thyrosine hydroxylase, Olfactory Marker Protein and cyclic AMP-dependent protein kinase as an intracellular pathway involved in dopaminergic neurotransmission. MPTP induced motor impairment, but no deficit in olfactory search. Thyrosine hydroxylase did not differ in olfactory bulb, while a strong decrease was detected in substantia nigra and tegmentum of MPTP mice. Olfactory Marker Protein decreased in the olfactory bulb of MPTP mice, while a cyclic AMP-dependent protein kinase increased in the inner granular layer of MPTP mice. Conclusions MPTP mice do not present behavioural deficits in olfactory search, yet immunoreactivity reveals modifications in the olfactory bulb, and suggests changes in intracellular signal processing, possibly linked to neuron survival after MPTP.

Guanosine 3',5'-monophosphate and Na or Ca ions affect the transmembrane movements of the same pool of intradiskal ions. Extradiskal Na ions activate the efflux of intradiskal Na ions. Extradiskal Ca ions activate the efflux of intradiskal Rb ions. Na and Ca ions activate Na/Ca or Ca/Ca exchange, as previously described. cGMP activates a membrane permeability for all the cations tested, as previously described. The reciprocal relations between cGMP and the other pathways for ion movements through disk membranes are systematically examined. Some analogies between the cGMP-activated permeability of the disk membranes and the light-sensitive conductance of the rod plasma outer membrane are discussed.

The high-affinity binding of the cGMP analogue 8-(5-thioacetamidofluorescein)-cGMP to rod outer segment membranes depleted of peripherally bound proteins has been defined by equilibrium dialysis (mean ± SD): (a) membranes contain about one cGMP binding site per 130 rhodopsin molecules; (b) the concentration of free ligand for half saturation is 2.0 ± 0.6 μM; (c) the apparent Hill coefficient of the bound versus free ligand relationship is 1.7 ± 0.5; (d) half saturation of the binding sites is sufficient for 85% activation of calcium permeability. A gating mechanism is proposed.

1. Puberty onset in female mice is accelerated by exposure to conspecific adult male urine, which acts through the vomeronasal organ and the accessory olfactory system. A distinctive component of adult male mouse urine is the major urinary protein complex (MUP), which is a lipocalin; it has a hydrophobic pocket that binds small endogenous volatile molecules. The MUP gene family also codes for a hexapeptide, which has four residues in common with the N-terminal region of MUP. 2. MUP, the volatiles bound to MUP and the MUP-related hexapeptide have been tested for the induction of puberty acceleration by measuring the increase in uterus weight related to the first pro-oestrus phase. MUP, together with its bound volatiles, induces puberty acceleration. Its activity is retained even when the volatiles have been removed either by organic extraction or competition displacement with a high-affinity ligand. 3. MUP-related hexapeptide also induces puberty acceleration in female mice. In contrast, the odorants bound to MUP do not exert this effect. 4. It is proposed that the vomeronasal organ contains receptors that recognize the short N-terminal consensus sequence, N-Glu-Glu-Ala-X-Ser (where X is a polar residue), common to both MUP and the hexapeptide.