Immunocytochemical evidence for intragranular processing of pro-opiomelanocortin in the melanotropic cells of the rabbit

Summary The immunogold technique, employing antisera with clear-cut specificities, was used to localise different processing stages of pro-opiomelanocortin (POMC) in rabbit melanotropic cells. While the antiserum against ₃-MSH labelled all the secretory granules including intrasaccular condensations in the Golgi apparatus, antisera against -MSH only labelled extra-Golgi secretory vesicles (SV). All extra-Golgi SV were likewise labelled with the three antisera against -MSH used, despite their different specificities for the desacetylated, N-acetylated or C-amidated forms of the peptide. The antibody against -endorphin also labelled the extra-Golgi SV, while only some SV were labelled with the antibody against -endorphin. These results correlate with biochemical data in favour of mainly — if not exclusively — intragranular processing of POMC. Except for ₃-MSH, the cleavage of which could coincide with Golgi packaging of secretory material, other post-translational modifications of the precursor seem to occur when SV are discharged outside the Golgi area. The cleavage of -endorphin appears to be a later step in POMC processing, occurring in some mature SV.     

Expression of neural cell adhesion molecules,NCAMs, and their polysialylated forms,PSA-NCAMs,in the developing rat pituitary gland

Neural cell adhesion molecules (NCAMs) can undergo post-translational modifications, such as the addition of polysialic acid chains, thus generating PSANCAMs, which are expressed mainly during development. Since polysialylation considerably modifies NCAM adhesivity, expression of NCAMs and PSANCAMs has been investigated in the developing hypophysis by immunohistochemistry. At embryonic day 13 (E13), an antibody against NCAM outlined all cellular profiles in the entire Rathke's pouch; this labelling persisted until adulthood. NCAM expression increased in all lobes during development and concerned all pituitary cell types. In contrast, at E13, PSA-NCAMs were only detected in the neural lobe, solely constituted of pituicytes at this stage, and the tuberal lobe, the only lobe expressing hormonal mRNA at the same stage. PSA-NCAMs expression increased in the neural lobe at E17 with the arrival of the neurosecretory fibres and persisted into adulthood. In the anterior lobe, PSA-NCAMs appeared at E15 where their distribution was similar to that of the differentiating corticotrophic cells; at subsequent stages, their expression extended to the whole anterior lobe. Only two cell types, corticotrophic and somatotrophic cells, remained labelled in the adult gland. In the intermediate lobe, melanotrophic cells never expressed PSA-NCAMs but these were expressed on folliculo-stellate cells at birth, preceding the onset of innervation. These results suggest that NCAMs and PSA-NCAMs play a role in pituitary histogenesis, cell differentiation and neurointermediate lobe innervation.     

Revisiting the localisation of Zn cations sorbed on pathological apatite calcifications made through X-ray absorption spectroscopy

The role of oligo-elements such as Zn in the genesis of pathological calcifications is widely debated in the literature. An essential element of discussion is given by their localisation either at the surface or within the Ca apatite crystalline network. To determine the localisation, X-ray absorption experiments have been performed at SOLEIL. The Exafs results suggest that Zn atoms, present in the Zn²⁺ form, are bound to about 4 O atoms at a distance of 2.00 Å, while the interatomic distance R_CaO ranges between 2.35 Å and 2.71 Å. Taking into account the content of Zn (around 1000 ppm) and the difference in ionic radius between Zn²⁺ (0.074 nm) and Ca²⁺ (0.099 nm), a significant longer interatomic distance would be expected in the case of Zn replacing Ca within the apatite crystalline network. We thus conclude that Zn atoms are localised at the surface and not in the apatite nanocrystal structure. Such structural result has essential biological implications for at least two reasons. Some oligoelements have a marked effect on the transformation of chemical phases, and may modify the morphology of crystals. These are both major issues because, in the case of kidney stones, the medical treatment depends strongly on the precise chemical phase and on the morphology of the biological entities at both macroscopic and mesoscopic scales.     

Calcium-sensitivity of the plasmalemmal delayed rectifier potassium current suggests that calcium influx in pulvinar protoplasts from Mimosa pudica L. can be revealed by hyperpolarization

Isolated protoplasts from pulvinar motor cells of Mimosa pudica were studied using conventional whole-cell patch clamp techniques. With internal solutions weakly buffered for Ca²⁺ (0.2 mm EGTA), a run-down of the outward delayed rectifier K⁺ current was induced by hyperpolarizing the holding potential, and this effect was strongly promoted by high external Ca²⁺ concentrations. This rundown could be reversed by coming back to less hyperpolarized holding potentials or by lowering the external [Ca²⁺]. Such rundown was absent when pipette internal solutions strongly buffered (10 mm EGTA) for Ca²⁺ were used. Ionomycin induced run-down of the K⁺ current with internal solutions containing 0.2 mm but not 10 mm EGTA. The hyperpolarization-associated rundown was reversibly blocked by Gd³⁺ and La³⁺.We thank Christophe Untereiner and Denis Wagner for expert technical assistance in facilitating the experiments and data acquisition and analysis.     

Colchicine effects on neurosecretory neurons and other hypothalamic and hypophysial cells,with special reference to changes in the cytoplasmic membranes

Summary The morphological effects of colchicine on the entire neurosecretory (NS) tract and on various hypothalamic nuclei have been studied. The perturbation in axonal flow, indicated by the accumulation of NS material, coincide with fragmentation of the cytoplasmic membranes, i. e. the Golgi apparatus and the endoplasmic reticulum, whereas the neurotubules remain relatively well preserved. Autophagic destruction of NS material is observed along the entire length of the NS fibres. The rapid and systematic changes in the axoplasmic reticulum, known to store calcium, lead us to envisage a role for this system — similar to that of the sarcoplasmic reticulum — in controlling the transport of NS vesicles. The junctional zone between the stalk and the neural lobe seems to play a particular rôle in the transport of NS material to the posthypophysial terminals of the NS axons. Colchicine provokes an increase in dense-cored vesicles in most of the neurons of the other hypothalamic nuclei studied: arcuate, suprachiasmatic, periventricular and ventromedial. Membranous alterations are also observed in these sites. Colchicine administered to animals which were hypothyroid, castrated or adrenalectomized, reveals stimulated neurons, identified by their excessive content of dense-cored vesicles. These neurons display no specific localization, for they occur in all hypothalamic nuclei, irrespective of the stimulation. The frequency of stimulation of neurons of the periventricular nucleus is striking.     

Effects of experimental hypo-or hypernatremia on the fine structure of the pars intermedia of the murine pituitary

Summary Quantified ultrastructural observations of the pars intermedia (PI) of the murine hypophysis enable evaluation and kinetic study of relatively fine secretory changes in the gland. Changes in volume of rER and newly formed dense secretory granules (Golgi granules) appear to best translate functional variations in the PI, as shown by the morphological effects of drugs affecting the dopaminergic control of the gland. Our morphometric results show that the PI is stimulated, but only briefly (no longer than 8–12 days), by both salt-loading and Na deprivation. However, the PI displays different secretory patterns in salt-loaded and Na-deprived mice; moreover, bromocriptine, which abolishes PI stimulation in Na-deprived mice, has only a slight inhibitory effect in salt-treated animals. Thus, it appears that the stimulation of the PI under both experimental conditions is triggered by different mechanisms. These results underline the plurifactorial control of the PI and show that the gland may have complex effects on hydromineral regulation.     

Adventitia-derived nitric oxide in rat aortas exposed to endotoxin: cell origin and functional consequences

The role of adventitial cells in bacterial lipopolysaccharide (LPS)-induced vascular nitric oxide (NO) overproduction has been largely ignored. In rat aortas exposed to LPS in vitro or in vivo, it was found that adventitia contained the major part of NO synthase (NOS)-2 protein (Western blot and immunohistochemistry) and generated the largest amount of NO (electron paramagnetic resonance spin trapping). NOS-2 immunoreactive cells were mainly resident macrophages at an early stage (5 h, in vitro or in vivo) and fibroblasts at a later stage (20 h, in vitro). Adventitial NOS-2 activity largely accounted for 1) the relaxing effect of L-arginine in rings exposed to LPS in vivo, 2) generation of an "NO store" revealed by N-acetylcysteine-induced relaxation, and 3) formation of protein-bound dinitrosyl iron complexes in the medial layer of aortic rings exposed to LPS in vitro. In conclusion, the adventitia is a powerful source of NO triggered by LPS in the rat aorta. This novel source of NO has an important impact on smooth muscle function and might be implicated in various inflammatory diseases.     

Microevolution of S-allele frequencies in wild cherry populations: respective impacts of negative frequency dependent selection and genetic drift

Negative frequency dependent selection (NFDS) is supposed to be the main force controlling allele evolution at the gametophytic self-incompatibility locus (S-locus) in strictly outcrossing species. Genetic drift also influences S-allele evolution. In perennial sessile organisms, evolution of allelic frequencies over two generations is mainly shaped by individual fecundities and spatial processes. Using wild cherry populations between two successive generations, we tested whether S-alleles evolved following NFDS qualitative and quantitative predictions. We showed that allelic variation was negatively correlated with parental allelic frequency as expected under NFDS. However, NFDS predictions in finite population failed to predict more than half S-allele quantitative evolution. We developed a spatially explicit mating model that included the S-locus. We studied the effects of self-incompatibility and local drift within populations due to pollen dispersal in spatially distributed individuals, and variation in male fecundity on male mating success and allelic frequency evolution. Male mating success was negatively related to male allelic frequency as expected under NFDS. Spatial genetic structure combined with self-incompatibility resulted in higher effective pollen dispersal. Limited pollen dispersal in structured distributions of individuals and genotypes and unequal pollen production significantly contributed to S-allele frequency evolution by creating local drift effects strong enough to counteract the NFDS effect on some alleles.