Mechanosensing role of caveolae and caveolar constituents in human endothelial cells

A variety of evidence suggests that endothelial cell functions are impaired in altered gravity conditions. Nevertheless, the effects of hypergravity on endothelial cell physiology remain unclear. In this study we cultured primary human endothelial cells under mild hypergravity conditions for 24-48 h, then we evaluated the changes in cell cycle progression, caveolin1 gene expression and in the caveolae status by confocal microscopy. Moreover, we analyzed the activity of enzymes known to be resident in caveolae such as endothelial nitric oxide synthase (eNOS), cycloxygenase 2 (COX-2), and prostacyclin synthase (PGIS). Finally, we performed a three-dimensional in vitro collagen gel test to evaluate the modification of the angiogenic responses. Results indicate that hypergravity shifts endothelial cells to G(0)/G(1) phase of cell cycle, reducing S phase, increasing caveolin1 gene expression and causing an increased distribution of caveolae in the cell interior. Hypergravity also increases COX-2 expression, nitric oxide (NO) and prostacyclin (PGI2) production, and inhibits angiogenesis as evaluated by 3-D collagen gel test, through a pathway not involving apoptosis. Thus, endothelial cell caveolae may be responsible for adaptation of endothelium to hypergravity and the mechanism of adaptation involves an increased caveolin1 gene expression coupled to upregulation of vasodilators as NO and PGI2.     

A polyamine analogue prevents acute pancreatitis and restores early liver regeneration in transgenic rats with activated polyamine catabolism

We recently generated a transgenic rat model for acute pancreatitis, which was apparently caused by a massive depletion of pancreatic polyamines spermidine and spermine due to inducible activation of their catabolism (Alhonen, L., Parkkinen, J. J., Kein?nen, T., Sinervirta, R., Herzig, K. H., and J?nne, J. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 8290-8295). When subjected to partial hepatectomy, these animals showed striking activation of polyamine catabolism at 24 h postoperatively with a profound decrease in hepatic spermidine and spermine pools and failure to initiate liver regeneration. Here we show that pancreatitis in this model could be totally prevented, as judged by histopathology and plasma alpha-amylase activity, by administration of 1-methylspermidine, a metabolically stable analogue of spermidine. Similarly, the analogue, given prior to partial hepatectomy, restored early liver regeneration in the transgenic rats, as indicated by a dramatic increase in the number of proliferating cell nuclear antigen-positive hepatocytes from about 1% to more than 40% in response to the drug. The present results suggest that the extremely high concentration of spermidine in the pancreas, in fact the highest in the mammalian body, may have a critical role in maintaining organ integrity. The failure to initiate liver regeneration in the absence of sufficient hepatic polyamine pools similarly indicates that polyamines are required for proper commencement of the regenerative process.     

Individual and seasonal variation in fecal testosterone and cortisol levels of wild male tufted capuchin monkeys, Cebus apella nigritus

This study tested the "challenge hypothesis" and rank-based predictions for temporal steroid production in male tufted capuchin monkeys, Cebus apella. Fecal samples (n = 209) collected from six wild males were analyzed for testosterone and cortisol concentration by enzyme immunoassay. The temporal pattern in male steroid production was compared to female sexual activity and rates of male aggression. The top-ranking adult male did not differ from other adult males in testosterone or cortisol concentration. Mean adult testosterone was significantly higher than mean subadult testosterone throughout the year. There was a clear elevation of testosterone and cortisol in both adult and subadult males during the peak of adult female sexual activity after the birth season. In fact, the magnitude of increase in testosterone was higher than predicted for a species with low male-male aggression. However, there was no difference between nonbreeding baseline testosterone levels during the birth season, and the "breeding" baseline of testosterone in males found during asynchronous female sexual activity. Of all behavioral indices examined, the distribution of female-maintained consortships was the best predictor of mean adult male testosterone concentrations. Although in many species, elevated testosterone coincides with increased male-male aggression, in the present study, the sustained high-magnitude increase in steroids during the peak of adult female sexual activity was associated with a relatively low rate of male-male intragroup aggression.     

p53-independent apoptosis in UV-irradiated mouse skin: possible inhibition by 50 Hz magnetic fields

Our recent results suggest that 50 Hz magnetic fields (MF) enhance ultraviolet (UV)-induced tumorigenesis in mouse skin. The aim of the present experiment was to study suppression of apoptosis as a possible mechanism for MF effects on skin tumorigenesis. Another aim was to test the importance of a UV and MF exposure schedule, particularly the role of MF exposure prior to UV irradiation. Female mice were exposed to a UV dose of 2 human MED and to 100 microT MF of 50 Hz, using the following exposure schedules: group 1 sham MF 24 h, UV 1 h, sham MF 24 h; group 2 sham MF 24 h, UV 1 h, MF 24 h; group 3 MF 24 h, UV 1 h, MF 24 h. Lamps emitting simulated solar radiation (SSR) were used for UV irradiation. Skin samples were analysed for apoptosis, expression of the p53 gene, activity of the enzyme ornithine decarboxylase (ODC) and polyamine concentrations. A significantly (p = 0.017) lower number of apoptotic cells was measured in group 2 compared to group 1. A similar but not statistically significant (p = 0.064) decrease was also detected in group 3. No p53 expression was detected in any sample. The levels of ODC and putrescine did not differ significantly between the UV-only and UV and MF-exposed groups. Spermidine and spermine levels were significantly (p = 0.014 and 0.014, respectively) lower in group 3 than in group 1, but no decrease was observed in group 2. Our findings suggest that SSR induces p53-independent apoptosis in mouse skin and that the apoptotic response may be inhibited by exposure to MF. The exposure schedule did not alter the MF effect. The results do not support a causal role for polyamines in MF effects on apoptosis.     

Disturbances in glucose-tolerance, insulin-release, and stress-induced hyperglycemia upon disruption of the Ca(v)2.3 (alpha 1E) subunit of voltage-gated Ca(2+) channels

Multiple types of voltage-activated Ca(2+) channels (T, L, N, P, Q, R type) coordinate Ca(2+)-dependent processes in neurons and neuroendocrine cells. Expressional and functional data have suggested a role for Ca(v)2.3 Ca(2+) channels in endocrine processes. To verify its role in vivo, Ca(v)2.3(-/-) mutant mice were generated, thus deficient in alpha 1E/R-type Ca(2+) channel. Intraperitoneal injection of D-glucose showed that glucose tolerance was markedly reduced, and insulin release into plasma was impaired in Ca(v)2.3-deficient mice. In isolated islets of Langerhans from these animals, no glucose-induced insulin release was detected. Further, in stressed Ca(v)2.3-deficient mice, the rate of glucose release into the blood was only 29% of that observed for wild-type animals. Thus, the deletion of Ca(v)2.3 causes deficits not only in insulin release but also in stress-induced hyperglycemia. The complex phenotype of Ca(v)2.3-deficient mice has dual components related to endocrine and neurological defects. The present findings provide direct evidence of a functional role for the Ca(v)2.3 subunit in hormone secretion and glucose homeostasis.     

Genes associated with the end of dormancy in grapes

A grape bud EST library was constructed and 4,270 ESTs sequenced. The library clones were arrayed for the purpose of investigating the level of gene expression over time, particularly leading up to the buds release from dormancy. The arrays were hybridized with P³³-labeled probes produced from samples of buds collected at weekly intervals. These probes covered the time from 9 weeks prior to bud burst until just after the emergence of the shoots. Expression patterns from these genes have been examined. It was found that 74% of the genes in the data set were homologous to known proteins. Genes were then assigned to functional categories according to their primary BLAST match. Of these 13% were involved with photosynthesis, 13% with disease resistance and defense, 5% energy, 12% metabolism, 20% protein production and processing, 25% cell structure and plant growth and the remaining 12% were unclassified The expression pattern of a selection of candidate genes retrieved from literature previously reporting an association with dormancy changes was assessed. On closer examination most of these genes relate to the oxidative processes and stress responses within the cell. The results of this study show that even in the dormant state, gene expression in the buds is high.     

Increased salt sensitivity secondary to leptin resistance in SHHF rats is mediated by endothelin

A link between leptin resistance, obesity, and salt sensitivity has been suggested. SHHF/Mcc-fa ^cp rats (SHHF) were used to study the effect of gene dosage of a null mutation of the leptin receptor (cp) on salt sensitivity and response to a combined endothelin A and B receptor antagonist (bosentan). Obese (cp/cp), heterozygous (+/cp), and homozygous lean (+/+) male SHHF were fed a low salt diet (0.3% NaCl) for 7 days, followed by a high salt diet (8.0% NaCl) for 7 days. There were no significant differences in systolic blood pressure between genotypes on low salt. In response to high salt, cp/cp had significantly greater systolic pressure than +/cp and +/+. On high salt diet, cp/cp showed a significant increase in 24 h urinary endothelin excretion and increased renal expression of preproendothelin mRNA. There was no effect of high salt diet on renal excretion of nitric oxide (NOx) or on gene expression of endothelial, neuronal, or cytokine-induced nitric oxide synthase isoforms (eNOS, nNOS, iNOS, respectively). Treatment with bosentan prevented the high salt-induced increment in systolic blood pressure in cp/cp. This was associated with a doubling of renal NOx excretion, but without changes in eNOS, nNOS, or iNOS expression. Endothelin receptor antagonism did not normalize systolic pressure in any of the genotypes. Our studies indicate that obesity secondary to leptin resistance (cp/cp) results in increased salt sensitivity that is mediated by endothelin in the SHHF rat.     

Functional specialization of presynaptic Cav2.3 Ca2+ channels

Ca2+ influx into presynaptic terminals via voltage-dependent Ca2+ channels triggers fast neurotransmitter release as well as different forms of synaptic plasticity. Using electrophysiological and genetic techniques we demonstrate that presynaptic Ca2+ entry through Cav2.3 subunits contributes to the induction of mossy fiber LTP and posttetanic potentiation by brief trains of presynaptic action potentials while they do not play a role in fast synaptic transmission, paired-pulse facilitation, or frequency facilitation. This functional specialization is most likely achieved by a localization remote from the release machinery and by a Cav2.3 channel-dependent facilitation of presynaptic Ca2+ influx. Thus, the presence of Cav2.3 channels boosts the accumulation of presynaptic Ca2+ triggering presynaptic LTP and posttetanic potentiation without affecting the low release probability that is a prerequisite for the enormous plasticity displayed by mossy fiber synapses.     

The role of flow cytometry in the study of cell growth in the rat anterior pituitary gland

Flow cytometry is a suitable technique for studying in vivo and in vitro the cell cycle kinetics of different animal and human tissues, both in normal and tumoral conditions. The rat anterior pituitary gland is a model to investigate cell growth and replication of differentiated, neuroendocrine cells, and we report current evidence on its cell cycle kinetics as well as on the role played by flow cytometry in this type of study. The proliferation potential of normal anterior pituitary cells is related to a number of different conditions, including heterogeneity of cell types, age and sex of donors, and circadian influences. In addition, the trend of cell proliferation in both in vivo and in vitro studies is similar, suggesting that cultured anterior pituitary elements may, at least in part, retain growth features analogous to those of the intact gland. Sorting of selective cell types and analysis of the relation between proliferating anterior pituitary cells and the light-dark cycle have shown that flow cytometry may be useful to investigate the replication process of the gland. By using a combination of flow cytometry, light microscopic immunocytochemistry and morphometry, we have reported a peculiar trend of proliferation in primary monolayer cultures of rat anterior pituitary gland, characterized by a non-linear reduction in their proliferation rate with advancing age, primarily dependent on a reduced transition of cells from the G0/G1- to the early S-phase pool. These studies indicate that flow cytometry offers insights into cell cycle check points of anterior pituitary cells, and suggest that it might be applied to the study of growth of selective pituitary elements, both in normal and tumoral conditions.