Assessment of climate change effects on mountain ecosystems through a cross-site analysis in the Alps and Apennines.

Mountain ecosystems are sensitive and reliable indicators of climate change. Long-term studies may be extremely useful in assessing the responses of high-elevation ecosystems to climate change and other anthropogenic drivers from a broad ecological perspective. Mountain research sites within the LTER (Long-Term Ecological Research) network are representative of various types of ecosystems and span a wide bioclimatic and elevational range. Here, we present a synthesis and a review of the main results from ecological studies in mountain ecosystems at 20 LTER sites in Italy, Switzerland and Austria covering in most cases more than two decades of observations. We analyzed a set of key climate parameters, such as temperature and snow cover duration, in relation to vascular plant species composition, plant traits, abundance patterns, pedoclimate, nutrient dynamics in soils and water, phenology and composition of freshwater biota. The overall results highlight the rapid response of mountain ecosystems to climate change, with site-specific characteristics and rates. As temperatures increased, vegetation cover in alpine and subalpine summits increased as well. Years with limited snow cover duration caused an increase in soil temperature and microbial biomass during the growing season. Effects on freshwater ecosystems were also observed, in terms of increases in solutes, decreases in nitrates and changes in plankton phenology and benthos communities. This work highlights the importance of comparing and integrating long-term ecological data collected in different ecosystems for a more comprehensive overview of the ecological effects of climate change. Nevertheless, there is a need for (i) adopting co-located monitoring site networks to improve our ability to obtain sound results from cross-site analysis, (ii) carrying out further studies, in particular short-term analyses with fine spatial and temporal resolutions to improve our understanding of responses to extreme events, and (iii) increasing comparability and standardizing protocols across networks to distinguish local patterns from global patterns.

Is phosphorylation of the alpha1 subunit at Ser-16 involved in the control of Na,K-ATPase activity by phorbol ester-activated protein kinase C?

The alpha1 subunit of Na,K-ATPase is phosphorylated at Ser-16 by phorbol ester-sensitive protein kinase(s) C (PKC). The role of Ser-16 phosphorylation was analyzed in COS-7 cells stably expressing wild-type or mutant (T15A/S16A and S16D-E) ouabain-resistant Bufo alpha1 subunits. In cells incubated at 37 degrees C, phorbol 12, 13-dibutyrate (PDBu) inhibited the transport activity and decreased the cell surface expression of wild-type and mutant Na,K-pumps equally ( approximately 20-30%). This effect of PDBu was mimicked by arachidonic acid and was dependent on PKC, phospholipase A(2), and cytochrome P450-dependent monooxygenase. In contrast, incubation of cells at 18 degrees C suppressed the down-regulation of Na,K-pumps and revealed a phosphorylation-dependent stimulation of the transport activity of Na,K-ATPase. Na,K-ATPase from cells expressing alpha1-mutants mimicking Ser-16 phosphorylation (S16D or S16E) exhibited an increase in the apparent Na affinity. This finding was confirmed by the PDBu-induced increase in Na sensitivity of the activity of Na,K-ATPase measured in permeabilized nontransfected COS-7 cells. These results illustrate the complexity of the regulation of Na,K-ATPase alpha1 isozymes by phorbol ester-sensitive PKCs and reveal 1) a phosphorylation-independent decrease in cell surface expression and 2) a phosphorylation-dependent stimulation of the transport activity attributable to an increase in the apparent Na affinity.

Insulin-induced stimulation of Na+,K(+)-ATPase activity in kidney proximal tubule cells depends on phosphorylation of the alpha-subunit at Tyr-10.

Phosphorylation of the alpha-subunit of Na+,K(+)-ATPase plays an important role in the regulation of this pump. Recent studies suggest that insulin, known to increase solute and fluid reabsorption in mammalian proximal convoluted tubule (PCT), is stimulating Na+,K(+)-ATPase activity through the tyrosine phosphorylation process. This study was therefore undertaken to evaluate the role of tyrosine phosphorylation of the Na+,K(+)-ATPase alpha-subunit in the action of insulin. In rat PCT, insulin and orthovanadate (a tyrosine phosphatase inhibitor) increased tyrosine phosphorylation level of the alpha-subunit more than twofold. Their effects were not additive, suggesting a common mechanism of action. Insulin-induced tyrosine phosphorylation was prevented by genistein, a tyrosine kinase inhibitor. The site of tyrosine phosphorylation was identified on Tyr-10 by controlled trypsinolysis in rat PCTs and by site-directed mutagenesis in opossum kidney cells transfected with rat alpha-subunit. The functional relevance of Tyr-10 phosphorylation was assessed by 1) the abolition of insulin-induced stimulation of the ouabain-sensitive (86)Rb uptake in opossum kidney cells expressing mutant rat alpha1-subunits wherein tyrosine was replaced by alanine or glutamine; and 2) the similarity of the time course and dose dependency of the insulin-induced increase in ouabain-sensitive (86)Rb uptake and tyrosine phosphorylation. These findings indicate that phosphorylation of the Na+,K(+)-ATPase alpha-subunit at Tyr-10 likely participates in the physiological control of sodium reabsorption in PCT.

Effect of cAMP on the activity and the phosphorylation of Na+,K(+)-ATPase in rat thick ascending limb of Henle.

BACKGROUND: In rat kidney medullary thick ascending limb of Henle's loop (MTAL), activation of protein kinase A (PKA) was previously reported to inhibit Na+,K(+)-ATPase activity. This is paradoxical with the known stimulatory effect of cAMP on sodium reabsorption. Because this inhibition was mediated by phospholipase A2 (PLA2) activation, a pathway stimulated by hypoxia, we evaluated the influence of oxygen supply on cAMP action on Na+,K(+)-ATPase in MTAL. METHODS: Ouabain-sensitive 86Rb uptake and Na+,K(+)-ATPase activity were measured in isolated MTALs. Cellular ATP content and the phosphorylation level of Na+,K(+)-ATPase were determined in suspensions of outer medullary tubules. Experiments were carried out under nonoxygenated or oxygenated conditions in the absence or presence of PKA activators. RESULTS: cAMP analogues or forskolin associated with 3-isobutyl-1-methylxanthine (IBMX) inhibited ouabain-sensitive 86Rb uptake in nonoxygenated MTALs. In contrast, when oxygen supply was increased, cAMP stimulated ouabain-sensitive 86Rb uptake and Na+,K(+)-ATPase activity. Improved oxygen supply was associated with increased intracellular ATP content. The phosphorylation level of the Na+,K(+)-ATPase alpha subunit was increased by cAMP analogues or forskolin associated with IBMX in oxygenated as well as in nonoxygenated tubules. Under nonoxygenated conditions, the inhibition of Na+,K(+)-ATPase was dissociated from its cAMP-dependent phosphorylation, whereas under oxygenated conditions, the stimulatory effect of cAMP analogues on ouabain-sensitive 86Rb uptake was linearly related and cosaturated with the level of phosphorylation of the Na+,K(+)-ATPase alpha subunit. CONCLUSION: In oxygenated MTALs, PKA-mediated stimulation of Na+,K(+)-ATPase likely participates in the cAMP-dependent stimulation of sodium reabsorption. Under nonoxygenated conditions, this stimulatory pathway is likely overridden by the PLA2-mediated inhibitory pathway, a possible adaptation to protect the cells against hypoxic damage.

Protein kinase A induces recruitment of active Na+,K+-ATPase units to the plasma membrane of rat proximal convoluted tubule cells.

1. The aim of this study was to investigate the mechanism of control of Na+,K+-ATPase activity by the cAMP-protein kinase A (PKA) pathway in rat proximal convoluted tubules. For this purpose, we studied the in vitro action of exogenous cAMP (10-3 M dibutyryl-cAMP (db-cAMP) or 8-bromo-cAMP) and endogenous cAMP (direct activation of adenylyl cyclases by 10-5 M forskolin) on Na+,K+-ATPase activity and membrane trafficking. 2. PKA activation stimulated both the cation transport and hydrolytic activity of Na+,K+-ATPase by about 40%. Transport activity stimulation was specific to the PKA signalling pathway since (1) db-cAMP stimulated the ouabain-sensitive 86Rb+ uptake in a time- and dose-dependent fashion; (2) this effect was abolished by addition of H-89 or Rp-cAMPS, two structurally different PKA inhibitors; and (3) this stimulation was not affected by inhibition of protein kinase C (PKC) by GF109203X. The stimulatory effect of db-cAMP on the hydrolytic activity of Na+,K+-ATPase was accounted for by an increased maximal ATPase rate (Vmax) without alteration of the efficiency of the pump, suggesting that cAMP-PKA pathway was implicated in membrane redistribution control. 3. To test this hypothesis, we used two different approaches: (1) cell surface protein biotinylation and (2) subcellular fractionation. Both approaches confirmed that the cAMP-PKA pathway was implicated in membrane trafficking regulation. The stimulation of Na+,K+-ATPase activity by db-cAMP was associated with an increase (+40%) in Na+, K+-ATPase units expressed at the cell surface which was assessed by Western blotting after streptavidin precipitation of biotinylated cell surface proteins. Subcellular fractionation confirmed the increased expression in pump units at the cell surface which was accompanied by a decrease (-30%) in pump units located in the subcellular fraction corresponding to early endosomes. 4. In conclusion, PKA stimulates Na+,K+-ATPase activity, at least in part, by increasing the number of Na+-K+ pumps in the plasma membrane in proximal convoluted tubule cells.

Modulation of Na+,K(+)-ATPase activity by a tyrosine phosphorylation process in rat proximal convoluted tubule.

1. In the rat kidney proximal convoluted tubule, epidermal growth factor and insulin have been reported to stimulate Na+ reabsorption. Because most of the effects of these growth factors are mediated by a process of tyrosine phosphorylation and Na+,K(+)-ATPase drives Na+ reabsorption, the influence of tyrosine kinases and tyrosine phosphatases on Na+,K(+)-ATPase activity located in the proximal convoluted tubule was evaluated. 2. Activation of receptor tyrosine kinases by epidermal growth factor and insulin stimulated ouabain-sensitive 86Rb+ uptake. The effects of epidermal growth factor and insulin were prevented by genistein, a tyrosine kinase inhibitor, but were unaffected by GF109203X, a protein kinase C inhibitor. 3. Inhibition of tyrosine phosphatases by orthovanadate (10(-7) and 10(-6)M) mimicked the effects of activation of receptor tyrosine kinases: stimulation of the ouabain-sensitive 86Rb+ uptake and of the hydrolytic activity of Na+,K(+)-ATPase under rate-limiting Na+ concentration, and absence of modification of the maximal activity (Vmax) of the enzyme. The effects of orthovanadate and insulin on the ouabain-sensitive 86Rb+ uptake were not additive. 4. The present results show that both activation of receptor tyrosine kinases and inhibition of tyrosine phosphatases stimulate the Na+,K(+)-ATPase activity through a common mechanism. Thus, a tyrosine phosphorylation process directly controls the Na+,K(+)-ATPase activity and contributes to the physiological control of water and solute reabsorption in the proximal convoluted tubule.

Stimulation of ouabain-sensitive 86Rb+ uptake and Na+,K+-ATPase alpha-subunit phosphorylation by a cAMP-dependent signalling pathway in intact cells from rat kidney cortex.

We investigated in intact cortical kidney tubules the role of PKA-mediated phosphorylation in the short-term control of Na+,K+-ATPase activity. The phosphorylation level of Na+,K+-ATPase was evaluated after immunoprecipitation of the enzyme from 32P-labelled cortical tubules and the cation transport activity of Na+,K+-ATPase was measured by ouabain-sensitive 86Rb+ uptake. Incubation of cells with cAMP analogues (8-bromo-cAMP, dibutyryl-cAMP) or with forskolin plus 3-isobutyl-1-methylxanthine increased the phosphorylation level of the Na+,K+-ATPase alpha-subunit and stimulated ouabain-sensitive 86Rb+ uptake. Inhibition of PKA by H-89 blocked the effects of dibutyryl-cAMP on both phosphorylation and 86Rb+ uptake processes. The results suggest that phosphorylation by PKA stimulates the Na+,K+-ATPase activity.

Protein kinase C-dependent phosphorylation of Na(+)-K(+)-ATPase alpha-subunit in rat kidney cortical tubules.

We have previously shown that, in oxygenated rat kidney proximal convoluted tubules (PCT), activation of protein kinase C (PKC) by phorbol 12,13-dibutyrate (PDBu) directly stimulates Na(+)-K(+)-adenosinetriphosphatase (ATPase) activity. PKC modulation of Na(+)-K(+)-ATPase activity by phosphorylation of its alpha-subunit was the postulated mechanism. The present study was therefore designed to investigate the relationship between PKC-mediated phosphorylation of the catalytic alpha-subunit and the cation transport activity of the Na(+)-K(+)-ATPase. In a suspension of rat kidney cortical tubules, activation of PKC by 10(-7) M PDBu increased the level of phosphorylation of the Na(+)-K(+)-ATPase alpha-subunit and stimulated the ouabain-sensitive 86Rb uptake by 47 and 42%, respectively. Time and dose dependence of the PDBu-induced increase in Na(+)-K(+)-ATPase activity and phosphorylation was strongly linearly correlated. The effects of PDBu on phosphorylation and activity of Na(+)-K(+)-ATPase were prevented by GF-109203X, a specific PKC inhibitor, whereas H-89, a specific PKA inhibitor, was ineffective. These results demonstrate that PKC activation induces phosphorylation of the catalytic alpha-subunit of Na(+)-K(+)-ATPase, which may participate in the stimulation of its cation transport activity in the rat PCT.

Presence of two isoforms of Na, K-ATPase with different pharmacological and immunological properties in the rat kidney.

Previous studies have demonstrated the presence of two populations of Na,K-ATPase with distinct kinetic, pharmacological and immunological characteristics along the rabbit nephron, indicating that the proximal segments of the nephron express exclusively the alpha 1 isoform of the catalytic subunit, whereas the collecting duct expresses an alpha 3-like isoform. Because pharmacological studies have shown the existence of two populations of Na,K-ATPase with different sensitivities to ouabain in the rat cortical collecting duct, which may result from the presence in the same nephron segment of the two isoforms demonstrated in the different segments of the rabbit nephron, the present study was undertaken to characterize the properties of Na,K-ATPase along the rat nephron. Results indicate that each segment of the rat nephron contains two subpopulations of Na,K-ATPase: a component highly sensitive to ouabain (IC50 approximately 5.10(-6) M) which is recognized by an anti-alpha 3 antibody and another moiety of lower affinity for ouabain (IC50 approximately 5.10(-4) M) which is recognized by an anti-alpha 1 antibody. Whether these two subpopulations correspond to different isoforms of the alpha subunit of Na,K-ATPase (alpha 1 and alpha 3-like) remains to be determined.

Protein kinase C-dependent stimulation of Na(+)-K(+)-ATP epsilon in rat proximal convoluted tubules.

In rat proximal convoluted tubule (PCT), activation of protein kinase C (PKC) by phorbol 12,13-dibutyrate (PDBu) was previously reported to inhibit Na(+)-K(+)-ATPase, a paradoxical finding in view of the known stimulatory effect of PKC on Na+ reabsorption. Because this inhibition occurs via phospholipase A2 activation, a pathway stimulated by hypoxia, we evaluated the influence of oxygen supply on PKC action on Na(+)-K(+)-ATPase. Results confirmed that PDBu inhibited PCT Na(+)-K(+)-ATPase activity under usual conditions. In contrast, when oxygen supply was increased, PDBu had no effect on Na(+)-K(+)-ATPase hydrolytic activity, but it dose-dependently stimulated ouabain-sensitive 86Rb+ uptake. This latter effect, which was abolished by PKC inhibitors, resulted from an increment of the Na+ sensitivity of Na(+)-K(+)-ATPase. Thus, in oxygenated rat PCTs, activation of PKC primarily stimulated Na(+)-K(+)-ATPase. This likely contributes to increase solute reabsorption. Inhibition of Na(+)-K(+)-ATPase was observed only under hypoxic conditions. It may represent an adaptation to protect PCTs against deleterious effects of hypoxia.

Insulin enhances sodium sensitivity of Na-K-ATPase in isolated rat proximal convoluted tubule.

Insulin has been shown to stimulate the rate of ouabain-sensitive 86Rb influx in the isolated rat proximal convoluted tubule (PCT). To study the mechanism of this activation of Na-K-adenosinetriphosphatase (Na-K-ATPase), we determined the actions of insulin on 1) the maximal activity (Vmax) of Na-K-ATPase hydrolytic activity; 2) the maximal rate of ouabain-sensitive 86Rb influx (after intracellular Na loading); 3) the rate of ouabain-sensitive 86Rb influx under conditions where intracellular Na concentration is rate limiting, either in the presence or in the absence of 5 x 10(-4) M amiloride and/or low extracellular Na concentration (3 mM); and 4) the Na sensitivity of the Na-K-ATPase hydrolytic activity. The maximal rates of Na-K-ATPase hydrolytic activity and of ouabain-sensitive 86Rb uptake were unchanged by insulin. In contrast, we confirmed that insulin enhanced 86Rb uptake (in peq.mm-1.min-1) in the absence of inhibitor of the Na/H exchanger [18.2 +/- 1.7 to 24.1 +/- 1.3 (SE), P < 0.03] and, in addition, demonstrated a similar stimulation in the presence of either 5 x 10(-4) M amiloride (7.2 +/- 0.6 to 10.7 +/- 0.9, P < 0.01), 3 mM extracellular Na (4.1 +/- 0.4 to 5.6 +/- 0.2, P < 0.05), and both amiloride and 3 mM extracellular Na (2.1 +/- 0.7 to 4.5 +/- 0.4, P < 0.03). Finally, insulin increased the sensitivity of Na-K-ATPase to Na as the apparent dissociation constant decreased from 46.5 +/- 5.3 to 27.6 +/- 3.0 mM (P < 0.03).(ABSTRACT TRUNCATED AT 250 WORDS)

[Compound complex odontoma (transitional): Report of a case]. / Odontoma compuesto complejo (de transición): informe de un caso.

This paper reports the case history of a nine-year old female referred to consultation because of clinical absence of left upper central incisor, with persistence of lateral incisor of homologous side, and with discrete volume increase of that region. Surgical procedure and post-op treatment are described, with confirmation, in this case, of clinical and histopathological diagnosis of complex-compound (transition) odontoma.