Fluorescence spectroscopic studies of pressure effects on Na+,K(+)-ATPase reconstituted into phospholipid bilayers and model raft mixtures.

To contribute to the understanding of membrane protein function upon application of pressure as relevant for understanding, for example, the physiology of deep sea organisms or for baroenzymological biotechnical processes, we investigated the influence of hydrostatic pressure on the activity of Na+,K+-ATPase enriched in the plasma membrane from rabbit kidney outer medulla using a kinetic assay that couples ATP hydrolysis to NADH oxidation. The data show that the activity of Na+,K+-ATPase is reversibly inhibited by pressures below 2 kbar. At higher pressures, the enzyme is irreversibly inactivated. To be able to explore the effect of the lipid matrix on enzyme activity, the enzyme was also reconstituted into various lipid bilayer systems of different chain length, conformation, phase state, and heterogeneity including model raft mixtures. To yield additional information on the conformation and phase state of the lipid bilayer systems, generalized polarization values by the Laurdan fluorescence technique were determined as well. Incorporation of the enzyme leads to a significant increase of the lipid chain order. Generally, similar to the enzyme activity in the natural plasma membrane, high hydrostatic pressures lead to a decline of the activity of the enzyme reconstituted into the various lipid bilayer systems, and in most cases, a multi-phasic behavior is observed. Interestingly, in the low-pressure region, around 100 bar, a significant increase of activity is observed for the enzyme reconstituted into DMPC and DOPC bilayers. Above 100-200 bar, this activity enhancement is followed by a steep decrease of activity up to about 800 bar, where a more or less broad plateau value is reached. The enzyme activity decreases to zero around 2 kbar for all reconstituted systems measured. A different scenario is observed for the effect of pressure on the enzyme activity in the model raft mixture. The coexistence of liquid-ordered and liquid-disordered domains with the possibility of lipid sorting in this lipid mixture leads to a reduced pressure sensitivity in the medium-pressure range. The decrease of ATPase activity may be induced by an increasing hydrophobic mismatch, leading to a decrease of the conformational dynamics of the protein and eventually subunit rearrangement. High pressures, above about 2.2 kbar, irreversibly change protein conformation, probably because of the dissociation and partial unfolding of the subunits.

Calcium transport in human salivary glands: a proposed model of calcium secretion into saliva.

Salivary calcium plays a vital role in bio-mineralization of dental enamel and exposed dentin. In order to elucidate the yet unknown cellular and molecular mechanisms of calcium secretion in human salivary glands the presence of various relevant plasma membrane transport systems for calcium were investigated. Using an RT-PCR approach, expression of the epithelial calcium channel (CaT-Like), the calcium binding protein (calbindin-2), the endoplasmic reticulum pumps (SERCA-2 and -3), and the plasma membrane calcium ATPases (PMCA-1, -2, and -4), were found in parotid and submandibular glands. Immunohistochemistry revealed that CaT-Like is located in the basolateral plasma membrane of acinar cells; while calbindin-2, SERCA-2 and SERCA-3 were found inside the acinar cells; and PMCA-2 was found in the apical membrane and in the secretory canaliculi between the cells. Based on these findings, we propose the following model of calcium secretion in human salivary glands: (1) calcium enters the acinar cell at the basolateral side via calcium channel CaT-Like (calcium influx); (2) intracellular calcium is taken up into the endoplasmic reticulum by SERCA-2 and possibly SERCA3 or bound to calbindin-2 (intracellular calcium pool); and (3) calcium is secreted by PMCAs at the apical plasma membrane (calcium efflux).

TMAO and other organic osmolytes in the muscles of amphipods (Crustacea) from shallow and deep water of Lake Baikal.

Concentrations of trimethylamine oxide (TMAO) and other 'compatible' osmolytes were analyzed in the muscle tissue of Lake Baikal amphipods (Crustacea) in relation to water depth of the freshwater Lake Baikal. Using HPLC and mass spectrometry, glycerophosphoryl choline (GPC), betaine, S-methyl-cysteine, sarcosine, and taurine were detected for the first time in freshwater amphipods. These osmolytes were frequently found in the five species studied but mixtures were too complex to be quantified. The pattern of these osmolytes did not change with respect to water depth. The TMAO concentration, however, was significantly higher in the muscle tissue of amphipods living in deep water than of those living in shallow water, which supports the hypothesis that TMAO acts as a protective osmolyte at increased hydrostatic pressure. We propose that eurybathic amphipods, exposed to raised hydrostatic pressure in the extremely deep freshwater Lake Baikal, have elevated TMAO levels to counteract the adverse effect of high pressure on protein structure. The elevated intracellular osmotic pressure is balanced by upregulating the extracellular hemolymph NaCl concentration.

Characterization of sulfate, proline, and glucose transport systems in anterior cruciate and medial collateral ligament cells.

The present study was undertaken to define the nature of key transport processes for sodium, glucose, proline, and sulfate in primary culture of canine anterior cruciate ligament (ACL) and medial collateral ligament (MCL) cells. Uptake studies using radiolabeled isotopes were performed and Na,K-ATPase activity was determined in cell lysates. At 25 degrees C both ACL and MCL cells showed a significant uptake of 86Rb. Ouabain inhibited Rb uptake by 55% in ACL cells and by 60% in MCL cells. The transport activity of Na,K-ATPase in intact cells was calculated to be 57 and 71 nmol.(mg protein)-1.(15 min)-1, respectively. The enzymatic activity of Na,K-ATPase in cell lysates was observed to be 104 for ACL cells and 121 nmol.(mg protein)-1.(15 min)-1 for MCL cells. Cytochalasin B, a known inhibitor of sodium-independent D-glucose transport, completely inhibited D-glucose uptake in ACL and MCL cells. Removal of Na+ or addition of 10-5 mol/L phlorizin, a potent inhibitor of the sodium-D-glucose cotransporter, did not alter D-glucose uptake, suggesting that glucose entered the cells using a sodium-independent pathway. Both ACL and MCL cells exhibited high sulfate uptake that was not altered by replacement of Na+ by N-methyl-D-glucamine, whereas DIDS, an inhibitor of sulfate/anion exchange abolished sulfate uptake in both cell types. Thus, neither cell type seems to possess a sodium-sulfate cotransport system. Rather, sulfate uptake appeared to be mediated by sulfate/anion exchange. Proline was rapidly taken up by ACL and MCL cells and its uptake was reduced by 85% when Na+ was replaced by N-methyl-D-glucamine, indicating that proline entered the cells via sodium-dependent cotransport systems. The data demonstrate that both ACL and MCL cells possess a highly active sodium pump, a secondary active sodium-proline cotransport system, and sodium-independent transport systems for D-glucose and sulfate.

Permeabilities of teleost and elasmobranch gill apical membranes: evidence that lipid bilayers alone do not account for barrier function.

Teleosts and elasmobranchs faced with considerable osmotic challenges living in sea water, use compensatory mechanisms to survive the loss of water (teleosts) and urea (elasmobranchs) across epithelial surfaces. We hypothesized that the gill, with a high surface area for gas exchange must have an apical membrane of exceptionally low permeability to prevent equilibration between seawater and plasma. We isolated apical membrane vesicles from the gills of Pleuronectes americanus (winter flounder) and Squalus acanthias (dogfish shark) and demonstrated approximately sixfold enrichment of the apical marker, ADPase compared to homogenate. We also isolated basolateral membranes from shark gill (enriched 2.3-fold for Na-K-ATPase) and using stopped-flow fluorometry measured membrane permeabilities to water, urea, and NH(3). Apical membrane water permeabilities were similar between species and quite low (7.4 +/- 0.7 x 10(-4) and 6.6 +/- 0.8 x 10(-4) cm/s for shark and flounder, respectively), whereas shark basolateral membranes showed twofold higher water permeability (14 +/- 2 x 10(-4) cm/s). Permeabilities to urea and NH(3) were also low in apical membranes. Because of the much lower apical to basolateral surface area we conclude that the apical membrane represents an effective barrier. However, the values we obtained were not low enough to account for low water loss (teleosts) and urea loss (elasmobranchs) measured in vivo by others. We conclude that there are other mechanisms which permit gill epithelia to serve as effective barriers. This conclusion has implications for the function of other barrier epithelia, such as the gastric mucosa, mammalian bladder, and renal thick ascending limb.