Regulation of the immunoexpression of aquaporin 9 by ovarian hormones in the rat oviductal epithelium

The volume of oviductal fluid fluctuates during the estrous cycle, suggesting that water availability is under hormonal control. It has been postulated that sex-steroid hormones may regulate aquaporin (AQP) channels involved in water movement across cell membranes. Using a functional assay (oocytes of Xenopus laevis), we demonstrated that the rat oviductal epithelium contains mRNAs coding for water channels, and we identified by RT-PCR the mRNAs for AQP5, -8, and -9, but not for AQP2 and -3. The immunoreactivity for AQP5, -8, and -9 was localized only in epithelial cells of the oviduct. The distribution of AQP5 and -8 was mainly cytoplasmic, whereas we confirmed, by confocal microscopy, that AQP9 localized to the apical plasma membrane. Staining of AQP5, -8, and -9 was lost after ovariectomy, and only AQP9 immunoreactivity was restored after estradiol and/or progesterone treatments. The recovery of AQP9 reactivity after ovariectomy correlated with increased mRNA and protein levels after treatment with estradiol alone or progesterone administration after estradiol priming. Interestingly, progesterone administration after progesterone priming also induced AQP9 expression but without a change in mRNA levels. Levels of AQP9 varied along the estrous cycle with their highest levels during proestrus and estrus. These results indicate that steroid hormones regulate AQP9 expression at the mRNA and protein level and that other ovarian signals are involved in the expression of AQP5 and -8. Thus hormonal regulation of the type and quantity of water channels in this epithelium might control water transport in the oviductal lumen. water channels; epithelial cells; estradiol; progesterone     

Distribution of peptidase activity in teleost and rat tissues

Peptides play important roles in cell regulation and signaling in many tissues. The actions of peptides are regulated by peptidases. Although the activity of these enzymes has been thoroughly characterized in mammals, little is known about their presence or function in fish. In the present study, we compared the activity of several peptidases in selected tissues (pituitary gland, different brain areas, kidney and gills) of the gilthead sea bream and rainbow trout with that found in similar rat tissues (lungs studied in place of gills). Soluble puromycin-sensitive aminopeptidase showed the highest values in the pituitary gland of the sea bream, whereas the membrane-bound form was found to be more active in the trout kidney. Very high levels of activity of aminopeptidase N were detected in trout and sea bream plasma. In contrast, the highest levels of activity of aminopeptidase B were found in rat tissues, with the exception of the gills of the trout. Aminopeptidase N levels tended to be higher in sea bream tissues with respect to those of trout. In contrast, the level of activity of aminopeptidase B was found to be consistently much higher in trout tissues than in those of the sea bream. Prolyl endopeptidase activity was principally detected in the pituitary gland and in the brain areas of teleosts. These differences between species could be related to different mechanisms of osmoregulation in saltwater- and in freshwater-adapted fish.     

Effects of Dihydrotestosterone on Brain mRNA Levels of Steroid 5α-Reductase Isozymes in Early Postnatal Life of Rat

We studied the expression of type 1 (5α-R1) and type 2 (5α-R2) 5α-reductase isozymes (5α-R) and their regulation by dihydrotestosterone (DHT) in the prefrontal cortex of male and female rats during postnatal sexual differentiation of the central nervous system (CNS), using one-step quantitative RT-PCR coupled with laser-induced fluorescence capillary electrophoresis. We found a higher expression of 5α-R2, which is considered a masculinizing enzyme, in the female versus male CNS, and observed sexual dimorphism in the regulation of both 5α-R isozymes by DHT. These results open up a new research line that could improve understanding of the role of 5α-R isozymes in the physiology of the CNS.     

Role of cAMP-Dependent Protein Kinase on Acute Picrotoxin-Induced Seizures

cAMP-dependent protein kinase (PKA) is a major modulator of synaptic transmission likely to be involved in molecular and cellular events leading to epileptogenesis, but little is known about how it affects the onset of acute epileptic seizures. In this study, we determined PKA enzymatic activity in the rat hippocampus during picrotoxin-induced seizures, using H-9 dihydrochloride, a PKA inhibitor, to investigate the in vivo effects of this enzyme on seizures induced by picrotoxin microdialysis in the rat hippocampus. No significant modifications were found in PKA activity during seizures as compared to control rats, but H-9 dihydrochloride microperfusion (100 μM) prevented picrotoxin seizures in 50% of the animals and significantly reduced the mean number of seizures and mean seizure duration. These results suggest that acute picrotoxin-induced seizures occur without an increase in hippocampal PKA activity, but reduced PKA-mediated phosphorylation protects against picrotoxin seizures, probably by increasing the inhibitory potential of GABA_A receptors. The possibility of other targets for H-9 dihydrochloride, such as PKC, PKG or CAMKII, however, cannot be ruled out.     

Cytological analysis of early microspore divisions leading to gametic embryo formation in <Emphasis Type="Italic">Quercus suber</Emphasis> L. anther cultures

The correlation between the phenologic stage of the inflorescence and the microspore development stage was studied. Cytological examinations of the development of microspores during in vitro anther culture of cork oak (Quercus suber L.), were carried out during the first four weeks of culture. To observe the division occurring in the microspores, anthers were taken randomly from the cultures after heat shock treatment and were stained with DAPI. Most of the anthers responding to a heat stress treatment contained 91 % vacuolated microspores, indicating that this developmental stage is responsive to embryogenesis induction in cork-oak microspores. After the heat shock treatment some cork-oak microspores were induced and initiated the embryogenic pathway with the occurrence of numerous symmetric mitosis, producing structures with two to ten or more nuclei. These lead to the formation of high numbers of multicellular cork-oak microspores (pro-embryos). Twenty-forty days after induction, small white globular and cotyledonal embryos were observed, which further developed root and shoot, regenerating plantlets.     

Cell architecture during gametophytic and embryogenic microspore development in <Emphasis Type="Italic">Brassica napus</Emphasis> L.

In this work, the cell architecture of the microspore following both gametophytic and embryogenic developmental pathways in vitro was compared with the gametophytic development in vivo in Brassica napus, at both light and electron microscopy level. The microspore reprogramming to embryogenesis involves defined changes affecting cell activities and structural organization which can be considered as markers of the microspore embryogenic pathway, but less is known about others developmental programmes followed by the microspore in vitro after both, inductive and non-inductive conditions. Low-temperature processing of the samples, cytochemical and immunocytochemical approaches to identify various cell components were performed. Differences in specific cellular features such as cellular size and shape, nuclear architecture, starch accumulation, presence of vacuoles and ribosomal population were studied to characterize sequential stages of microspore embryogenesis and other pathways occurring in vitro. The presence of abundant starch grains in a defined cytoplasmic region appeared as a specific feature of the in vitro gametophytic development, as well as of the non-induced microspores of in vitro cultures under embryogenic-inductive conditions.     

Variation in Dispersal Traits in a Narrow-endemic Plant Species, <Emphasis Type="Italic">Centaurea corymbosa</Emphasis> Pourret. (Asteraceae)

The existence of genetic variability for dispersal is a crucial issue for organisms facing increased habitat fragmentation and climate change. We study the genetic basis and evolutionary potential for diaspore traits related to dispersal in Centaurea corymbosa. Using diaspores collected in natural conditions in four of the six extant populations of this narrow-endemic plant species and diaspores produced in a common garden experiment, we study the variation for pappus and achene sizes, and diaspore mass. Using a sample of achenes from the common garden experiment, we find that the best predictor of terminal velocity is a linear combination of pappus length, achene width, and achene weight. We find significant differences among populations for all traits in both conditions, as well as significant differences among families within population. Although the differences among populations for some traits are not exactly the same in controlled conditions compared to natural conditions, the ranking of populations according to their mean trait values is consistent in both conditions. Our study is therefore one of the first to show a correlation between phenotypic differentiation for dispersal traits in natural conditions vs. controlled conditions. We also show evidence of genetic variation for traits commonly thought to be involved in dispersal ability, suggesting the potential for evolutionary changes following environmental change and management actions.Co-ordinating editor: J.F. Stuefer     

Role of Nitric Oxide on Motor Behavior

The present review paper describes results indicating the influence of nitric oxide (NO) on motor control. Our last studies showed that systemic injections of low doses of inhibitors of NO synthase (NOS), the enzyme responsible for NO formation, induce anxiolytic effects in the elevated plus maze whereas higher doses decrease maze exploration. Also, NOS inhibitors decrease locomotion and rearing in an open field arena.These results may involve motor effects of this compounds, since inhibitors of NOS, NG-nitro-L-arginine (L-NOARG), N^G-nitro-L-arginine methylester (L-NAME), N^G-monomethyl-L-arginine (L-NMMA), and 7-Nitroindazole (7-NIO), induced catalepsy in mice. This effect was also found in rats after systemic, intracebroventricular or intrastriatal administration.Acute administration of L-NOARG has an additive cataleptic effect with haloperidol, a dopamine D2 antagonist. The catalepsy is also potentiated by WAY 100135 (5-HT1a receptor antagonist), ketanserin (5HT2a and alfa1 adrenergic receptor antagonist), and ritanserin (5-HT2a and 5HT2c receptor antagonist). Atropine sulfate and biperiden, antimuscarinic drugs, block L-NOARG-induced catalepsy in mice.L-NOARG subchronic administration in mice induces rapid tolerance (3 days) to its cataleptic effects. It also produces cross-tolerance to haloperidol-induced catalepsy. After subchronic L-NOARG treatment there is an increase in the density NADPH-d positive neurons in the dorsal part of nucleus caudate-putamen, nucleus accumbens, and tegmental pedunculupontinus nucleus. In contrast, this treatment decreases NADPH-d neuronal number in the substantia nigra compacta.Considering these results we suggest that (i) NO may modulate motor behavior, probably by interfering with dopaminergic, serotonergic, and cholinergic neurotransmission in the striatum; (ii) Subchronic NO synthesis inhibition induces plastic changes in NO-producing neurons in brain areas related to motor control and causes cross-tolerance to the cataleptic effect of haloperidol, raising the possibility that such treatments could decrease motor side effects associated with antipsychotic medications.Finally, recent studies using experimental Parkinsons disease models suggest an interaction between NO system and neurodegenerative processes in the nigrostriatal pathway. It provides evidence of a protective role of NO. Together, our results indicate that NO may be a key participant on physiological and pathophysiological processes in the nigrostriatal system.     

H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced changes in mitochondrial activity in isolated mouse pancreatic acinar cells

This study employed confocal laser scanning microscopy to monitor the effect of H₂O₂ on cytosolic as well as mitochondrial calcium (Ca²⁺) concentrations, mitochondrial inner membrane potential (_m) and flavine adenine dinucleotide (FAD) oxidation state in isolated mouse pancreatic acinar cells. The results show that incubation of pancreatic acinar cells with H₂O₂, in the absence of extracellular Ca²⁺ ([Ca²⁺]_o) led to an increase either in cytosolic and in mitochondrial Ca²⁺ concentration. Additionally, H₂O₂ induced a depolarization of mitochondria and increased oxidized FAD level. Pretreatment of cells with the mitochondrial inhibitors rotenone or cyanide inhibited the response induced by H₂O₂ on mitochondrial inner membrane potential but failed to block oxidation of FAD in the presence of H₂O₂. However, the H₂O₂-evoked effect on FAD state was blocked by pretreatment of cells with the mitochondrial uncoupler, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP). On the other hand, perfusion of cells with thapsigargin (Tps), an inhibitor of the SERCA pump, led to an increase in mitochondrial Ca²⁺ concentration and in oxidized FAD level, and depolarized mitochondria. Pretreatment of cells with thapsigargin inhibited H₂O₂-evoked changes in mitochondrial Ca²⁺ concentration but not those in membrane potential and FAD state. The present results have indicated that H₂O₂ can evoke marked changes in mitochondrial activity that might be due to the oxidant nature of H₂O₂. This in turn could represent the mechanism of action of ROS to induce cellular damage leading to cell dysfunction and generation of pathologies in the pancreas. (Mol Cell Biochem 269: 165–173, 2005)