The Peculiar Facets of Nitric Oxide as a Cellular Messenger: From Disease-Associated Signaling to the Regulation of Brain Bioenergetics and Neurovascular Coupling

In this review, we address the regulatory and toxic role of ^·NO along several pathways, from the gut to the brain. Initially, we address the role on ^·NO in the regulation of mitochondrial respiration with emphasis on the possible contribution to Parkinson’s disease via mechanisms that involve its interaction with a major dopamine metabolite, DOPAC. In parallel with initial discoveries of the inhibition of mitochondrial respiration by ^·NO, it became clear the potential for toxic ^·NO-mediated mechanisms involving the production of more reactive species and the post-translational modification of mitochondrial proteins. Accordingly, we have proposed a novel mechanism potentially leading to dopaminergic cell death, providing evidence that NO synergistically interact with DOPAC in promoting cell death via mechanisms that involve GSH depletion. The modulatory role of NO will be then briefly discussed as a master regulator on brain energy metabolism. The energy metabolism in the brain is central to the understanding of brain function and disease. The core role of ^·NO in the regulation of brain metabolism and vascular responses is further substantiated by discussing its role as a mediator of neurovascular coupling, the increase in local microvessels blood flow in response to spatially restricted increase of neuronal activity. The many facets of NO as intracellular and intercellular messenger, conveying information associated with its spatial and temporal concentration dynamics, involve not only the discussion of its reactions and potential targets on a defined biological environment but also the regulation of its synthesis by the family of nitric oxide synthases. More recently, a novel pathway, out of control of NOS, has been the subject of a great deal of controversy, the nitrate:nitrite:NO pathway, adding new perspectives to ^·NO biology. Thus, finally, this novel pathway will be addressed in connection with nitrate consumption in the diet and the beneficial effects of protein nitration by reactive nitrogen species.

     

Cyclo(His-Pro) up-regulates heme oxygenase 1 via activation of Nrf2-ARE signalling

Paraquat (1,1'-dimethyl-4,4'-bipyridinium), a widely used non-selective herbicide, is a redox cycling agent with adverse effects on dopamine systems. Epidemiological data have shown that exposure to paraquat is one of the several risk factors for Parkinson's disease. We have already shown that cyclo(His-Pro), an endogenous cyclic dipeptide produced by the cleavage of the thyrotropin releasing hormone, has a cytoprotective effect through a mechanism involving Nrf2 activation that decreases production of reactive oxygen species and increases glutathione synthesis. Using primary neuronal cultures and PC12 cells as targets of paraquat neurotoxicity, we addressed whether and how cyclo(His-Pro) causes cellular protective response against paraquat-mediated cell death. We found that cyclo(His-Pro) attenuated reactive oxygen species production, and prevented glutathione depletion by up-regulating Nrf2 gene expression, triggering its nuclear accumulation and activating the expression of heme oxygenase1. These protective effects were abolished by RNA interference-mediated Nrf2 knock down whereas were unaffected by RNA interference-mediated Keap1 knock down. Inhibition of heme oxygenase activity decreased cyclo(His-Pro)-induced neuroprotection. These results suggest that cyclo(His-Pro), acting as a selective activator of the brain modulable Nrf2 pathway, may be a promising candidate as neuroprotective agent that act through induction of phase II genes.     

Oligomeric State and Thermal Stability of Apo- and Holo- Human Ornithine δ-Aminotransferase

Human ornithine δ-aminotransferase (hOAT) (EC 2.6.1.13) is a mitochondrial pyridoxal 5′-phosphate (PLP)-dependent aminotransferase whose deficit is associated with gyrate atrophy, a rare autosomal recessive disorder causing progressive blindness and chorioretinal degeneration. Here, both the apo- and holo-form of recombinant hOAT were characterized by means of spectroscopic, kinetic, chromatographic and computational techniques. The results indicate that apo and holo-hOAT (a) show a similar tertiary structure, even if apo displays a more pronounced exposure of hydrophobic patches, (b) exhibit a tetrameric structure with a tetramer-dimer equilibrium dissociation constant about fivefold higher for the apoform with respect to the holoform, and (c) have apparent T_m values of 46 and 67?°C, respectively. Moreover, unlike holo-hOAT, apo-hOAT is prone to unfolding and aggregation under physiological conditions. We also identified Arg217 as an important hot-spot at the dimer–dimer interface of hOAT and demonstrated that the artificial dimeric variant R217A exhibits spectroscopic properties, T_m values and catalytic features similar to those of the tetrameric species. This finding indicates that the catalytic unit of hOAT is the dimer. However, under physiological conditions the apo-tetramer is slightly less prone to unfolding and aggregation than the apo-dimer. The possible implications of the data for the intracellular stability and regulation of hOAT are discussed.     

Long‐term population dynamics of a coral reef gastropod and responses to disturbance

The age‐specific density of the red‐lipped stromb Strombus luhuanus (Mollusca: Gastropoda) was monitored over 13 years (1981–1993) at four locations on the intertidal reef flat at Heron Island, Great Barrier Reef. Densities were highly variable, but there were persistent, location‐specific differences in population density, age structure and adult body size, the latter indicating that the populations were not extensively linked by adult movement. There was relatively high recruitment at most locations in 1984, 1989 and 1993, each occurring approximately 2 years after El Niño/Southern Oscillation events, although recruit density during these years varied in both space and time. The studied strombs experienced three disturbance events: (i) experimental harvesting at two locations (1984–1985); (ii) siltation from a harbour dredging operation (1987–1988); and (iii) a severe cyclone (1992). Resilience to harvesting at a local scale (0.5–2 ha) was high: density had recovered within a year, due to immigration of adults and older juveniles. Strombus luhuanus responded much more strongly to broad‐scale changes to its environment than to localized harvesting. After dredging, there was a progressive density decline coupled with low recruitment at two locations, and a later decline at a third location, followed by a recruitment‐driven rebound after the cyclone. Generalized environmental effects of siltation and the cyclone were also reflected in substantial changes in algal cover. Long‐term variations in environmental conditions probably cause high temporal variation over large spatial scales through effects on the survival of larvae or recruits. Localized short‐term field monitoring of such species would give a misleading picture of key factors affecting population dynamics.     

Synthesis, conformation, and biological activity of the carbohydrate-free vespulakinin 1

Synthesis of the carbohydrate-free heptadecapeptide corresponding to the amino acid sequence of vespulakinin 1 was achieved by the continuous flow solid phase procedure on 4-hydroxymethyl-phenoxyacetyl-norleucyl derivatized Kieselguhr-supported polydimethylacrylamide resin, as well as by a combination of solid phase and solution syntheses. Preformed Fmoc-amino acid symmetrical anhydrides (Boc derivative for the N-terminal residue) were used for amine acylation in the continuous flow method. Serine and threonine were side chain protected as tert.-butyl ethers and the 4-methoxy-2, 3, 6,-trimethyl-benzenesulfonyl group was used for masking the guanidino function of arginine residues. After cleavage from the resin the final peptide was purified by ion exchange chromatography and characterized by amino acid analysis, high voltage electrophoresis, and RP-HPLC analysis. Alternatively, the protected N-terminal octapeptide, Fmoc-Thr(But)-Ala-Thr(But)-Thr(But)-Arg(Mtr)-Arg-(Mtr)-Arg(Mtr)-Gly-OH was prepared on 4-hydroxymethyl-3-methoxyphenoxyacetyl-norleucyl derivatized Kieselguhr-supported polydimethylacrylamide resin and the C-terminal nonapeptide H-Arg(NO2)-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-(NO2)-OBzl was synthesized in solution through the fragment condensation method. The two fragments were coupled by the DCC-HOBt procedure and the resulting heptadecapeptide was deblocked and purified. The conformational features of the synthesized peptides are reported. Preliminary pharmacological experiments indicated that carbohydrate-free vespulakinin 1 is more potent than bradykinin in lowering rat blood pressure.     

Chromosomal aberrations induced in human cultured cells by liposome-encapsulated deoxyribonuclease I

Experiments of incorporation of a nucleolytic enzyme into human cells cultured in vitro have been carried out with the aim of inducing structural chromosome variations. Human heteroploid cells, either as asynchronous populations or enriched in mitoses, and PHA-stimulated lymphocytes were used as recipients. We found that all these cells when exposed to pancreatic DNAase I encapsulated in liposomes, either of multilamellar (MLV) or of small unilamellar (SUV) type, show an incidence of chromosome damage higher than that induced by the enzyme free in the incubation buffer. Our results indicate that liposomes are suitable vehicles for the transfer of an exogenous nuclease into human cultured cells. The enzyme remains functionally active and interacts with nuclear DNA, giving rise to chromosome lesions.     

Synthesis and biological activity of tuftsin and rigin derivatives containing monosaccharides or monosaccharide derivatives

Synthesis of some modified rigins is described in which either D-gluconic acid or 2-amino-2-deoxy-beta-D-glucopyranose have been linked to the parent molecule through amide bonds involving the alpha-amino function, alpha-carboxyl function or the gamma-amide function of glutamine in position 2. Glu2-rigin and D-gluconyl-Glu2-rigin have also been synthesized. Binding and phagocytosis assays have been carried out on the rigin derivatives and on some glycosylated tuftsin derivatives as well. Of all the tested peptides only rigin enhanced the phagocytic capacity of mouse peritoneal macrophages to the same extent as tuftsin. The peptides H-Thr-Lys-Pro-Arg-NH-Glc and N alpha-gluconyl-Gly-Glu-Pro-Arg-OH slightly enhanced phagocytosis. H-Thr[(alpha + beta)-O-glucosyl]-Lys-Pro-Arg-OH was found to displace 3H-tuftsin even better than tuftsin but lacked the ability to stimulate phagocytosis.     

A transient voltage-dependent outward current in cultured cerebellar granules

Granule cells were dissociated from rat cerebella with a procedure that yields a 98% pure cell population. Potassium currents in these cells were studied using the patch-clamp technique. Depolarizing pulses of 10 mV step and 100 ms duration from a holding potential of –80 mV elicited two different potassium outward currents: a transient, low-voltage activated component and a long lasting, high-voltage activated component. At +30 mV, the total current reached an amplitude of 2 nA (mean value of 15 experiments). The reversal potential of the transient current, estimated by measuring tail currents, was –77 mV, close to that predicted by the Nernst equation. The transient current was half inactivated with a holding potential of –78 mV and completely inactivated with –50 mV or more positive holding potentials. Finally, the current decay could be fitted by the sum of two exponentials with time constants of about 20 and 250 ms.