Differential effects of eicosapentaenoic acid and docosahexaenoic acid on human skin fibroblasts

To better understand the mode of action of omega 3 fatty acids in cell membranes, human foreskin fibroblasts were grown in serum-free medium supplemented with 50 microM oleic acid linoleic acid, eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), and the effects on membrane composition, fluorescence polarization and enzyme activities were followed. The cells were enriched with EPA and DHA up to 7 and 13% of total lipids, respectively, of which > 95% was associated with phospholipids. In addition, the concentration of 22:5n-3 increased with both EPA and DHA to 7.5, and 2.1% of the total fatty acids, respectively. When compared to controls (oleic acid), cells treated with DHA showed a decrease in cholesterol, phospholipids, arachidonic acid (AA) and free cholesterol/phospholipid ratio (P < 0.05). In the presence of EPA, only decreases in AA and cholesterol were significant (P < 0.05). Membrane fluidity, assessed by fluorescence anisotropy, was increased 16% in cells enriched with DHA (P < 0.05), but showed no change with EPA or linoleic acid. There was an increase in membrane-associated 5'-nucleotidase (+27%) and adenylate cyclase (+19%) activities (P < 0.05), in DHA-enriched, but not in EPA-enriched cells, when compared with oleate controls. The studies show that incorporation of DHA, but not EPA, into cell membranes of fibroblasts alters membrane biophysical characteristics and function. We suggest that these two major n-3 fatty acids of fish oils have differential effects on cell membranes, and this may be related to the known differences in their physiological effects.     

稀土元素铈对红豆杉细胞膜透性的影响

用EvansBlue染色、细胞培养液电导率测定、MDA含量检测、电镜技术研究Ce4 对悬浮培养的东北红豆杉细胞膜的影响。结果表明:1mmol/LCe4 诱导后第5天,EvansBlue染色结果、细胞培养液的电导率、MDA含量显著地增加,细胞膜透性明显增大,细胞膜的完整性也受到一定程度的破坏,透射电镜观察细胞超微结构发生变化,说明1mmol/LCe4 能导致悬浮培养的红豆杉细胞膜的损伤。     

Acute fracture of the ulna occurring in a professional pitcher

AIM: To study the protective effects of tetrandrine (Tet) on CCl4-injured hepatocytes. METHODS: The cultured rat liver cells were poisoned by CCl4 (10 mmol.L-1). The membrane fluidity was detected by 1,6-diphenyl-1,3,5-hexatriene (DPH), a lipid probe. The Ca2+ concentration was assayed with Fura 2-AM, a sensitive calcium indicator. RESULTS: Tet (1-1000 nmol.L-1) increased viability of liver cell (from 71% to 72%-89%), reduced lactate dehydrogenase (LDH) release, and malondialdehyde (MDA) formation. Tet prevented the heightening of the intracellular Ca2+ concentration and the attenuation of the membrane fluidity of liver cells (P < 0.05). CONCLUSION: Tet had a protective effect on CCl4-injured hepatocytes by inhibiting the lipid peroxidation, improving the membrane fluidity, and lessening the Ca2+ concentration.     

Intracellular volumes and membrane permeability in rat hearts during prolonged hypothermic preservation with St Thomas and University of Wisconsin solutions

The study aims to determine a possible relationship between intracellular water, energy metabolism, functional recovery and membrane permeability, during and after hypothermic cardiac preservation. Isolated rat hearts were stored for 12 h at 4 degrees C with University of Wisconsin (UW), St Thomas Hospital (ST) and Krebs-Henseleit (KH) solutions, and were reperfused for 1 h. Cellular volumes were measured by 1H NMR of water and 59Co NMR of the extracellular marker cobalticyanide, and energetic profiles by 31P NMR spectroscopy. Storage in ST solution reduced ischemic swelling from 2.50 +/- 0.06 to 2.73 +/- 0.09 (P < 0.001 v 3.56 +/- 0.10 ml/g dry weight in KH), while UW solution caused cellular shrinkage to 2.12 +/- 0.08 ml/g dry weight. Intracellular ATP concentrations and pH values were higher in UW as compared to ST solution. At reperfusion, hearts stored in ST shrank while those stored in UW expanded, resulting in similar intracellular volumes. Storage with UW was superior to ST in post-ischemic function 65 +/- 5% (P < 0.01 v 49 +/- 4% with ST) and in recovery of ATP 46 +/- 3% (P < 0.001 v 25 +/- 4% with ST). Storage with both ST and UW solutions did not prevent interstitial edema. Sarcolemmal membrane integrity, as assessed by cellular swelling in response to a hypo-osmotic shock (210 mmol/l), was significantly improved by ST and UW solutions as compared to KH (P < 0.05). Creatine kinase efflux was reduced by ST and UW as compared to KH (P < 0.05), and by UW as compared to ST (P < 0.05). Coronary flow was higher following storage with UW than ST solutions. 66 +/- 6 and 45 +/- 4%, respectively (P < 0.01). According to these data, the beneficial effects of UW and ST solutions on hypothermic ischemic storage of rat hearts included prevention of cellular edema and preservation of sarcolemmal membrane integrity. It is concluded: (a) UW and ST solutions reduce ischemic and reperfusion cellular volumes: (b) both solutions, and UW in particular were efficient in preservation of membrane integrity: (c) prevention of cellular edema is not the single or main mechanism responsible for the improved preservation with UW and ST solutions.     

The presence of substituents on the aryl moiety of the aryl phosphoramidate derivative of d4T enhances anti-HIV efficacy in cell culture: A structure-activity relationship

Since thiols can undergo nitrosation and the cell membrane is rich in thiol-containing proteins, we considered the possibility that membrane surface thiols may regulate cellular entry of NO. Recently, protein disulfide isomerase (PDI), a protein that catalyzes thio-disulfide exchange reactions, has been found on the cell-surface membrane. We hypothesized that cell-surface PDI reacts with NO, catalyzes S-nitrosation reactions, and facilitates NO transfer from the extracellular to intracellular compartment. We observed that PDI catalyzes the S-nitrosothiol-dependent oxidation of the heme group of myoglobin (15-fold increase in the rate of oxidation compared with control), and that NO reduces the activity of PDI by 73.1 +/- 21.8% (P < 0.005). To assess the role of PDI in the cellular action of NO, we inhibited human erythroleukemia (HEL) cell-surface PDI expression using an antisense phosphorothioate oligodeoxynucleotide directed against PDI mRNA. This oligodeoxynucleotide decreased cell-surface PDI content by 74.1 +/- 9.3% and PDI folding activity by 46.6 +/- 3.5% compared with untreated or "scrambled" phosphorothioate oligodeoxynucleotide-treated cells (P < 0.0001). This decrease in cell-surface PDI was associated with a significant decrease in cyclic guanosine monophosphate (cGMP) generation after S-nitrosothiol exposure (65.4 +/- 26.7% reduction compared with control; P < 0.05), with no effect on cyclic adenosine monophosphate (cAMP) generation after prostaglandin E1 exposure. These data demonstrate that the cellular entry of NO involves a transnitrosation mechanism catalyzed by cell-surface PDI. These observations suggest a unique mechanism by which extracellular NO gains access to the intracellular environment.     

KATP channel modulators and myocardial damages induced by ischemia-reperfusion: membrane lipids injury and arrhythmias

Although KATP channels have been proposed as playing a role in most types of myocardial damage associated with ischemia/reperfusion, the potential benefits of KATP channel modulators against the biochemical and electrical disturbances observed during ischemia remain unclear. We have thus studied the effects of glibenclamide and cromakalim, KATP channel blocker and opener respectively, on membrane lipid injury and arrhythmias, in a model of ischemic-reperfused guinea-pig myocardium. Ventricular strips were prelabeled with [3H] arachidonic acid, then subjected to normal conditions (Time-related Control) or to simulated ischemic-reperfused conditions in absence of drug (Control) or in presence of glibenclamide 10 microM or cromakalim 10 microM. The release of radioactive compounds was counted by liquid scintillation spectrometry, while action potentials (AP) were recorded with intracellular microelectrodes. Reperfusion induced a significant increase of arachidonic acid release (P < 0.05 versus Time-related Control). Glibenclamide inhibited the reperfusion-induced arachidonic acid release while cromakalim only delayed it (respectively 483 +/- 87 dpm/g, P < 0.05 and 790 +/- 143 dpm/g. NS versus 838 +/- 80 dpm/g for Control, after 30 min of reperfusion). Unlike glibenclamide, cromakalim was proarrhythmic during reperfusion (in 100% of preparations versus 33% in Control or in presence of glibenclamide, P < 0.05). This in vitro study shows that glibenclamide prevented the reperfusion-induced membrane arachidonic acid release, without proarrhythmic effect, whereas cromakalim, associated with proarrhythmicity, was unable to protect myocardium from cell lipid damage.     

Altered retinoblastoma protein expression in nonsmall cell lung cancer: its synergistic effects with altered ras and p53 protein status on prognosis

BACKGROUND: Inactivation of the retinoblastoma (Rb) gene has been documented in various types of cancer, including lung cancer. Alterations of the p53 and ras genes are also common features in the molecular biology of lung carcinoma, and the authors of this article have reported previously on the prognostic significance of both of them. In the present study, the authors evaluated the prognostic significance of the loss of Rb protein expression alone, then performed a combined analysis of Rb protein and ras p21 status (Rb/ras) as well as an analysis of Rb and p53 protein status (Rb/p53) in patients with nonsmall cell lung cancer (NSCLC). METHODS: Ninety-one patients with NSCLC underwent potentially curative resection between 1977 and 1988, 65 of whom received postoperative combination chemotherapy. Tumor specimens were analyzed for Rb protein expression by immunohistochemistry. Univariate and multivariate analyses were performed to assess the association between Rb protein expression and survival. RESULTS: Nineteen (21%) of the 91 NSCLCs showed negative Rb protein expression. Positive or negative Rb protein expression (Rb+ or Rb-) as an individual factor was not statistically correlated with survival or prognosis in this cohort of NSCLC patients, although a tendency among Rb- patients to do worse was observed. The authors then combined the Rb protein status with previously studied results of ras p21 and p53 protein expression in the same tumor specimens, and compared the prognosis between the individuals with theoretically the best pattern of gene expression in their tumors and those with theoretically the worst pattern of expression, i.e., Rb+/ras- versus Rb-/ras+ and Rb+/p53- versus Rb-/p53+. In patients with adenocarcinoma, those with Rb-/ras+ tumors survived for a significantly shorter period after surgery (13% 5-year survival) than those with Rb+/ras- tumors (82% 5-year survival) (P = 0.01). Similarly, patients with Rb-/p53+ tumors survived for a significantly shorter period (20% 5-year survival) compared with those who had Rb+/p53- tumors (73% 5-year survival) (P = 0.008). Rb/ras status was a significant prognostic factor (P = 0.02 by univariate analysis, P = 0.048 by multivariate analysis), and Rb/p53 status tended to be significant as a prognostic factor (P = 0.04 by univariate analysis, P = 0.08 by multivariate analysis). In patients with squamous cell carcinoma, neither Rb/ras nor Rb/p53 status was a significant prognostic factor in this cohort. CONCLUSIONS: These results suggest that combined immunohistochemical analyses of Rb and ras p21 proteins and of Rb and p53 proteins may indicate their potentially synergistic effects on survival and prognosis. These analyses may also be useful for stratifying patients with adenocarcinoma of the lung into different prognostic groups and identifying populations with different risks of recurrence. Larger prospective studies with Stage I NSCLC patients are necessary to confirm the current findings.     

The human myoepithelial cell is a natural tumor suppressor

Myoepithelial cells, which surround ducts and acini of glandular organs, form a natural border separating proliferating epithelial cells from basement membrane and underlying stroma. Myoepithelial cells in situ and in vitro constitutively express high amounts of proteinase inhibitors that include tissue inhibitor of metalloproteinase 1, protease nexin-II, alpha-1 antitrypsin, and maspin. Human myoepithelial xenografts (HMS-X, HMS-3X, and HMS-4X), which our laboratory has established, accumulate an abundant extracellular matrix containing sequestered proteinase inhibitors. Humatrix, a gel that we have derived from HMS-X, inhibits tumor cell invasion (down to 25% +/- 10% of Matrigel control; P < 0.01), and our recently established human myoepithelial cell lines, HMS-1, HMS-3, and HMS-4, inhibit tumor cell invasion in cellular invasion (down to 42% +/- 7% of control; P < 0.05) and in conditioned media assays (down to 30% +/- 8% of control; P < 0.01). The anti-invasive effects of HMS-1, HMS-3, and HMS-4 can be enhanced by phorbol 12-myristate 13-acetate (down to 2% +/- 1% of control) by a maspin-dependent mechanism and abolished by dexamethasone (up to 95% +/- 5% of control) by a maspin-independent mechanism (P < 0.01). HMS-X, HMS-3X, HMS-4X, and Humatrix inhibit tumor invasion and metastasis in severe combined immunodeficient mice (P < 0.001). The cumulative data suggest that myoepithelial cells are natural paracrine suppressors of invasion and metastasis and may specifically inhibit the progression of precancerous disease states to invasive cancer in vivo.     

Osmolality and potassium cause alterations in the volume of glomerulosa cells

Alterations in extracellular osmolality have a powerful inverse effect on aldosterone secretion and potassium- and angiotensin-stimulated aldosterone secretion. Whether alterations in extracellular osmolality produced sustained changes in cell volume that may contribute to the regulation of aldosterone secretion is not known. Using dispersed bovine glomerulosa cells grown in primary culture, the effect of alterations in osmolality on cell volume, measured by the distribution of [14C]urea and [3H]inulin and videometric analysis of the surface area of glomerulosa cells, was determined. Alterations in osmolality had an inverse effect on cell volume and surface area. Changes in cell volume induced by exposure to anisotonic medium were 52% greater (P > 0.02) than that predicted by the changes in osmolality. Increases in potassium concentration also caused sustained (1-h) concentration-dependent increases in cell volume and surface area. Angiotensin-II did not increase glomerulosa cell volume, but did produce a small dose-dependent transient increase in cell surface area. The results demonstrate that alterations in osmolality do cause sustained changes in cell volume, and thus, membrane stretch could be an important part of the cellular mechanism responsible for causing osmolality-induced changes in the cytosolic calcium concentration and subsequent alterations in aldosterone secretion. Alterations in membrane stretch may also be an important component of potassium-induced, but not angiotensin II-induced, aldosterone secretion.     

Alpha(1)- and beta-adrenoceptor-mediated increase in 86Rb(+)-uptake in isolated cardiomyocytes from adult rat heart: evidence for interaction between the two receptor systems

The aim of the present investigation was to study the effect of alpha(1)- and beta-adrenoceptor stimulation, alone and in combination, on potassium uptake in isolated ventricular cardiomyocytes from adult rat heart, using the potassium analogue 86Rb+. The reliability of 86Rb+ as a potassium analogue was also investigated. Alpha(1)-, beta-, And combined adrenoceptor stimulation was achieved by using noradrenaline in the presence and absence of appropriate adrenoceptor antagonists. The uptake of 86Rb+ was found to increase linearly with time up to 20 min., both during basal and receptor-stimulated conditions. The basal uptake rate was about 0.18 ml/g protein x min. At 15 min. both alpha(1)-, beta- and combined adrenoceptor stimulation dose-dependently increased the 86Rb(+)-uptake with a -logEC50 of 7.05, 6.68 and 6.73, respectively. The maximal increase in these series achieved by 5 x 10(-5) mol/l noradrenaline was 29%, 24% and 41% above basal level, respectively. Comparison of the maximal effects in the same cell preparations, with the observed value for combined adrenoceptor stimulation in each experiment as 100%, gave a relative maximal increase in 86Rb(+)-uptake after separate alpha(1)-adrenoceptor stimulation of 67 +/- 8%, and of 68 +/- 6% after separate beta-adrenoceptor stimulation. The theoretically calculated value for combined adrenoceptor stimulation, if additivity, was 135 +/- 11%, which was significantly higher than the observed value (100%) (P = 0.026). The effect of noradrenaline was not limited by the maximal 86Rb(+)-uptake capacity, as 10(-5) mol/l forskolin increased the 86Rb(+)-uptake more than noradrenaline. Examining the reliability of 86Rb+ as potassium-analogue by combining 42K+ and 86Rb+ in the same experiments, showed that combined adrenoceptor stimulation dose-dependently increased both the 42K(+)- and 86Rb(+)-uptake with the same potency and to the same extent. Thus 86Rb+ is a reliable potassium-analogue for these effects. In conclusion both alpha(1)- and beta-adrenoceptor stimulation dose-dependently increased the cellular 86Rb(+)-uptake to the same extent and with the same potency. The observed maximal 86Rb(+)-uptake after combined adrenoceptor stimulation was significantly higher than the maximal effect after either form of separate receptor stimulation, but significantly lower than expected if the effects were purely additive. The results thus show inhibitory interaction between the two receptor systems.     

An SH2 domain-containing 5' inositolphosphatase inhibits insulin-induced GLUT4 translocation and growth factor-induced actin filament rearrangement

Tyrosine kinase receptors lead to rapid activation of phosphatidylinositol 3-kinase (PI3 kinase) and the subsequent formation of phosphatidylinositides (PtdIns) 3,4-P2 and PtdIns 3,4, 5-P3, which are thought to be involved in signaling for glucose transporter GLUT4 translocation, cytoskeletal rearrangement, and DNA synthesis. However, the specific role of each of these PtdIns in insulin and growth factor signaling is still mainly unknown. Therefore, we assessed, in the current study, the effect of SH2-containing inositol phosphatase (SHIP) expression on these biological effects. SHIP is a 5' phosphatase that decreases the intracellular levels of PtdIns 3,4,5-P3. Expression of SHIP after nuclear microinjection in 3T3-L1 adipocytes inhibited insulin-induced GLUT4 translocation by 100 +/- 21% (mean +/- the standard error) at submaximal (3 ng/ml) and 64 +/- 5% at maximal (10 ng/ml) insulin concentrations (P < 0.05 and P < 0.001, respectively). A catalytically inactive mutant of SHIP had no effect on insulin-induced GLUT4 translocation. Furthermore, SHIP also abolished GLUT4 translocation induced by a membrane-targeted catalytic subunit of PI3 kinase. In addition, insulin-, insulin-like growth factor I (IGF-I)-, and platelet-derived growth factor-induced cytoskeletal rearrangement, i.e., membrane ruffling, was significantly inhibited (78 +/- 10, 64 +/- 3, and 62 +/- 5%, respectively; P < 0.05 for all) in 3T3-L1 adipocytes. In a rat fibroblast cell line overexpressing the human insulin receptor (HIRc-B), SHIP inhibited membrane ruffling induced by insulin and IGF-I by 76 +/- 3% (P < 0.001) and 68 +/- 5% (P < 0.005), respectively. However, growth factor-induced stress fiber breakdown was not affected by SHIP expression. Finally, SHIP decreased significantly growth factor-induced mitogen-activated protein kinase activation and DNA synthesis. Expression of the catalytically inactive mutant had no effect on these cellular responses. In summary, our results show that expression of SHIP inhibits insulin-induced GLUT4 translocation, growth factor-induced membrane ruffling, and DNA synthesis, indicating that PtdIns 3,4,5-P3 is the key phospholipid product mediating these biological actions.     

Ginsenosides increase secretion of lipoprotein lipase by 3T3-L1 adipocytes

Treatment of 3T3-L1 adipocytes with either an oleanolic acid glycoside or a 20(S)-protopanaxatriol glycoside increased the secretion of lipoprotein lipase activity into the medium dose-dependently. At a concentration of 100 micrograms/ml, ginsenosides Ro, Re, Rg1, and Rh1 increased the secretion of lipase activity into the medium by 119, 107, 56, and 32%, respectively. The ratio of lipase activity in the medium to cellular lipase activity was 4.7% in control cells and 8.6% in ginsenoside Ro-treated cells, 8.3% in ginsenoside Re-treated cells, 7.0% in ginsenoside Rg1-treated cells, and 6.3% in ginsenoside Rh1-treated cells. Ginsenoside Rb2, which is a 20(S)-protopanaxadiol glycoside, increased the secretion of lipase activity by 16% at 25 micrograms/ml, and the ratio of lipase activity in the medium to cellular lipase activity was higher in ginsenoside Rb2-treated cells than in control cells. However, at 100 and 200 micrograms/ml, ginsenoside Rb2 decreased the secretion of lipase activity in parallel with cellular lipase activity. Ginsenoside Rd also decreased the secretion of lipase activity in the same dose-dependent manner. Thus, the effective dose for the secretion of lipoprotein lipase activity with ginsenosides varies with their aglycone structure.     

Heat shock- and ethanol-induced ionic changes in C6 rat glioma cells determined by NMR and fluorescence spectroscopy

The effects of two different stressors, heat shock (HS; 44 degrees C, 20 min) and ethanol (1.2 M, 60 min), on ion content and membrane potential were investigated in C6 rat glioma cells. Both treatments were previously shown to induce the HS response [26]. Intracellular pH (pH(i)), sodium ion concentration ([NA+]i), potassium ion concentration ([K+]i) and membrane potential were determined by means of continuous 31P and 23Na nuclear magnetic resonance (NMR), continuous fluorescence spectroscopy and 86Rb uptake. Lactate extrusion was determined in addition with respect to pH(i) regulation. The aim of this study was a detailed picture of HS and ethanol-induced ion changes in a single cell type, because stress-induced changes in the intracellular ionic balance may be important factors for determining proliferation, stress response and apoptosis. HS lowered the pH(i) from 7.38 +/- 0.04 to about 7.05 +/- 0.04. [Na+]i decreased during HS to 50% of the control and recovered to normal level 95 min after HS treatment. During HS, [K+]i remained constant but increased after HS. The membrane potential hyperpolarized from -83 mV to -125 mV and returned to initial values during HS treatment. Lactate extrusion increased 3-fold after HS. Ethanol (1.2 M) lowered the pH(i) from pH 7.38 +/- 0.04 to pH 7.0 +/- 0.04, but in contrast to heat strongly increased [Na]i. It hyperpolarized the membrane potential from -83 to -125 mV. Ethanol also increased lactate extrusion similar to HS. Also in contrast to the effect of HS, the potassium concentration decreased during ethanol treatment. The Na(+)-H+ exchanger monensin was used to overcome the apparent inhibition of the cellular Na(+)-H+ exchanger by HS. At normal pH(e) (7.4) monensin increased [Na+]i and pH(i) considerably. A subsequent HS reduced [Na+]i only minimally. Acidification of the cells by low pH(e) (6.2) prior to HS did not abolish the HS-induced drop of pH(i), indicating that the Na(+)-H+ exchanger was also inhibited at low pH(i). At low pH(e), monensin transports H+ into the cell. A subsequent HS decreased pH(i) only little, showing the importance of inhibition of the Na(+)-H+ exchanger for the HS-induced pH(i) decrease. 100 microM amiloride reduced pH(i) and [Na+]i in a similar way as HS, but did not change pH(i) and [Na+]i much during a HS. These results indicate that some of the HS-induced ionic changes are mediated by inhibition of the Na(+)-H+ exchanger, activation of Na(+)-K(+)-ATPase and changes of membrane conductance for ions.     

Self-assembled alpha-hemolysin pores in an S-layer-supported lipid bilayer

The mammalian distal colon, which is composed of different cell types, actively transports Na, K and Cl in absorptive and K and Cl in secretory directions. To further characterize the K absorption process and to identify the cells involved in K absorption, unidirectional Rb fluxes and luminal Rb uptake into different epithelial cell types were determined in isolated guinea-pig distal colon. Net Rb absorption (1.5-2.5 micromol.h-1.cm-2) was not influenced by inhibition of Na transport with amiloride or by incubating both sides of the epithelium with Na-free solutions, but was almost completely abolished by luminal ouabain, ethoxzolamide or by incubating both sides of the epithelium with Cl-free solutions. Luminal Rb uptake, blockable by luminal ouabain, preferentially occurred in columnar surface and neck cells, to a lesser extent in surface goblet cells and to an insignificant degree in lower crypt cells. Employing a luminal Rb-Ringer (5.4 mM Rb) the Rb concentration increased within 10 min in columnar surface and neck, surface goblet and lower crypt cells to 70, 32 and about 10 mmol. kg-1 wet weight, respectively. The presence of 5.4 mM K in the luminal incubation solution reduced Rb uptake almost completely indicating a much higher acceptance of the luminal H-K-ATPase for K than for Rb. The increase in Na and decrease in K concentrations in surface and neck cells induced by luminal ouabain might indicate inhibition of the basolateral Na-K-ATPase or drastic enhancement of cellular Na uptake by the Na-H exchanger. Bilateral Na-free incubation did not alter Rb uptake, but bilateral Cl-free incubation drastically reduced it. Inhibition of net Rb absorption by ethoxzolamide and inhibition of both Rb absorption and Rb uptake by bilateral Cl-free incubation support the notion that cellular CO2 hydration is a necessary prerequisite for K absorption and that HCO3 leaves the cell via a Cl-HCO3 exchanger. Since ouabain-inhibitable transepithelial Rb flux and luminal Rb uptake rate by surface and neck cells were about the same, Rb(K) absorption seems to be accomplished mainly by columnar surface cells.     

Taurine regulation of Ca2+ uptake and (Ca(2+)+Mg2+)-ATPase in developing chick B-cells

1. The objective of the present study was to determine the effect of age and taurine on chick B cell calcium uptake and membrane (Ca(2+)+Mg2+)-ATPase activity in 1-4-week-old chicks. 2. The calcium uptake rate decreased with age (P < 0.05) and was further decreased by taurine (P < 0.05). 3. (Ca(2+)+Mg2+)-ATPase activity increased with age (P < 0.05) and was stimulated by taurine (P < 0.05). 4. The data demonstrate that the flux of calcium across the B-cell membrane changes during early post-hatch development, and that taurine regulates both the influx and efflux of calcium in chick B-cells.     

Effects of bioabsorbable and non-resorbable barrier membranes on bone augmentation in rabbit calvaria

The aim of this study was to compare the effects of bioabsorbable and non-resorbable membranes on experimental guided bone augmentation in 8 Japanese white rabbits. A cutaneous flap was demarcated and raised from the forehead of each animal, the periosteum was lifted, and the calvarial bone on both sides of the midline was exposed. A titanium screw was inserted into the bone on each side of the midline and one screw was covered with a bioabsorbable (polylactic acid) membrane and the other with a non-resorbable (expanded polytetrafluoroethylene) membrane. The implanted screws and membranes were then covered with the periosteum and cutaneous flap. After healing for 6 months, the animals were euthanized and the experimental area was prepared for histological investigation. New bone had formed under both membranes with no sign of infection or membrane exposure. The amount of newly generated bone (89.0 +/- 17.3% versus 54.7 +/- 14.0%, P <0.05) and the percentage of newly generated bone height (81.5 +/- 6.3% versus 58.9 +/- 7.8%, P <0.05) in the space beneath the non-resorbable membrane was greater than that beneath the bioabsorbable membrane. However, there were no statistically significant differences between the bioabsorbable and non-resorbable membranes with respect to the percentage areas of mineralized bone (52.3 +/- 11.3% versus 47.1 +/- 6.7%, P = 0.8658) and bone marrow (47.7 +/- 11.3% versus 52.9 +/- 6.7%, P = 0.4838) and bone contact with the screw (88.3 +/- 6.9% versus 89.2 +/- 7.3%, P = 0.9999). In conclusion, at least within the limitations of this rabbit model, we suggest that non-resorbable membranes with sufficient stiffness should be used to obtain greater bone volume and height instead of bioabsorbable membranes for the GBR procedure, and that this will facilitate predictable bone augmentation in spaces beyond the bone surface. Therefore, the bioabsorbable membrane could not replace the non-resorbable membrane used in this model.     

Effect of fexofenadine on eosinophil-induced changes in epithelial permeability and cytokine release from nasal epithelial cells of patients with seasonal allergic rhinitis

Recent studies have suggested that antihistamines, widely used in the treatment of symptoms of patients with allergic rhinitis, may also possess antiinflammatory properties. The mechanisms underlying this property, however, are not clearly understood. We have cultured epithelial cells from nasal biopsy specimens from patients with seasonal allergic rhinitis outside the pollen season and studied the effect of 0 to 10(-3) mol/L fexofenadine, the main active metabolite of terfenadine, on eosinophil-induced changes in electrical resistance (measure of permeability) and release of proinflammatory mediators from these cells. Additionally, we have studied the effect of this drug on eosinophil chemotaxis and adherence to endothelial cells induced by conditioned medium from these human nasal epithelial cell (HNEC) cultures. Incubation of HNEC in the presence of eosinophils treated with opsonized latex beads significantly decreased the electrical resistance of these cultures, an effect that was abrogated by treatment of the cultures with 10(-9) to 10(-3) mol/L fexofenadine. Similarly, incubation of HNEC in the presence of eosinophils treated with latex beads also significantly increased the basal release of the chemokine "regulated upon activation, normal T cell expressed and secreted" (RANTES) (from 96.0 to 613.0 fg/microg cellular protein; p < 0.05), IL-8 (from 42.0 to 198.5 pg/microg cellular protein; p < 0.05), granulocyte-macrophage colony-stimulating factor (GM-CSF) (from 0.54 to 3.4 pg/microg cellular protein; p < 0.05), and soluble intercellular adhesion molecule-1 (sICAM-1) (from 7.8 to 18.4 pg/microg cellular protein; p < 0.05) from HNEC. The eosinophil-induced release of IL-8, GM-CSF, and sICAM-1 from the HNEC was significantly attenuated by treatment with fexofenadine. Analysis of the effects of conditioned medium from HNEC demonstrated that this significantly increased both eosinophil chemotaxis and adherence to endothelial cells. Addition of 10(-6) to 10(-3) mol/L fexofenadine to the conditioned medium significantly attenuated eosinophil chemotaxis and adherence to endothelial cells. These results suggest that fexofenadine may reduce nasal inflammation by modulating the release of proinflammatory mediators and adhesion molecules from HNEC.     

Interaction of Escherichia coli heat-stable enterotoxin B with cultured human intestinal epithelial cells

Binding of Escherichia coli heat-stable enterotoxin B (STb) to the human intestinal epithelial cell lines T84 and HT29 and to polarized T84 cells was studied to define the initial interaction of this peptide toxin with target cells. Equilibrium and competitive binding isotherms showed that 125I-STb bound specifically to T84 and HT29 cells; however, the toxin-epithelial cell interactions could be characterized by low-affinity binding (< or = 10(5) M(-1)) to a high number of binding sites (> or = 10(6) per cell). STb binding to T84 and HT29 cells as a function of 125I-STb concentration did not approach saturation at levels well above the effective biological concentration of STb for fluid secretion. Treatment of the 125I-STb-bound T84 and HT29 cells with an acidic saline solution to remove surface-bound toxin revealed that only approximately 55% +/- 10% of 125I-STb could be removed by this treatment at 4 degrees C, suggesting that approximately half of the bound STb was stably associated with the plasma membrane and/or internalized into the cytoplasm. Similar results were obtained when binding and internalization experiments were conducted at 22 and 37 degrees C. Immunofluorescence studies demonstrated that the strongest signal for STb appeared in the plasma membrane even after acid treatment. Toxin-treated cells also displayed diffuse cytoplasmic staining, indicating that once cell bound, STb did not appear to preferentially associate with membrane vesicles or cellular organelles. Binding and subsequent internalization of 125I-STb were not affected by treatment of the cells with trypsin, endoglycosidase F/peptide N-glycosidase F, Vibrio cholerae neuraminidase, tunicamycin, or 5 mM sodium chlorate, which blocks sulfation of surface proteoglycans. In addition, the internalization process was not altered by preincubation of the cells with the cytoskeleton inhibitors cytochalasin D and colchicine or cellular perturbants (i.e., 0.45 M sucrose and 5 mM sodium azide), indicating that cell surface proteins or carbohydrates did not function as STb receptors. The binding of 125I-STb to polarized T84 cells was also examined, and the total and nonspecific binding isotherms were found to overlap, indicating that the apical surface of polarized T84 cells did not contain a specific receptor for STb. In comparison to undifferentiated cells, twice the amount of bound STb (approximately 80% +/- 10%) was removable from polarized T84 cells after treatment with acidic solution. The percentage of surface-bound STb to polarized T84 cells did not vary significantly with the transepithelial electrical resistance of the cells or when STb was applied basolaterally. Together, our results indicate that STb binds with relatively low affinity to the plasma membrane of cultured intestinal epithelial cells and polarized T84 cells, probably to membrane lipids, and becomes stably associated with the lipid bilayer. The fact that a significant portion of the bound STb becomes free in the cytoplasm, even at a low temperature, suggests that the bound toxin may directly traverse the membrane bilayer.     

Elevated and absent pRb expression is associated with bladder cancer progression and has cooperative effects with p53

Rb protein (pRb) expression was evaluated in 185 cases of transitional cell carcinoma of the bladder from patients that underwent radical cystectomy. Tumors were stratified into three categories based on the percentage of nuclei expressing pRb: (a) 0, 0% of tumor cells showing nuclear reactivity; (b) 1+, 1-50% of tumor cells showing nuclear reactivity; and (c) 2+, >50% of tumor cells showing nuclear reactivity. Cases with undetectable (pRb 0) and high (pRb 2+) pRb reactivity had identical rates of recurrence. These cases had significantly higher recurrence (P = 0.0001) and lower survival rates (P = 0.0002) compared to cases with moderate (pRb 1+) pRb reactivity, indicating that high levels of pRb expression may reflect a dysfunctional (altered) Rb pathway. The tumors were also examined for alterations in p53 expression; patients with tumors altered in both p53 and pRb had significantly increased rates of recurrence (P < 0.0001) and survival (P < 0.0001) compared to patients with no alterations in either p53 or pRb; patients with alterations in only one of these proteins had intermediate rates of recurrence and survival. These results suggest that: (a) bladder cancers with high pRb expression do not show the tumor suppressor effects of the protein; and (b) alteration in both p53 and pRb may act in cooperative or synergistic ways to promote tumor progression.     

Regional deposition and retention of particles in shallow, inhaled boluses: effect of lung volume

We evaluated the effects of volatile anesthetics on T-type calcium current (ICa,T) present in four different cell types using the whole cell version of the patch clamp technique. In dorsal root ganglion neurons and in two neuroendocrine cells--adrenal glomerulosa cells (AG) and thyroid C-cells--ICa,T was reversibly decreased by volatile anesthetics at clinically relevant concentrations, with isoflurane and enflurane being more potent that halothane. In AG cells, the most sensitive cell type tested, ICa,T was reduced 47%+/-4% (n = 6) by isoflurane (0.7 mM) and 56%+/-2% (n = 5) by enflurane (1.2 mM), but by only 24%+/-1% (n = 5; P < 0.05) by halothane (0.7 mM). Isoflurane caused a significant increase in the rate of deactivation of ICa,T in AG cells. In ventricular myocytes, however, ICa,T was much less sensitive to both isoflurane and halothane. The differential sensitivity of ICa,T in various cell types to the anesthetics may reflect differences in the channels expressed in these tissues or differences in the cellular intermediates involved in anesthetic action. Depression of ICa,T in neuronal cells may contribute to anesthetic action through decreases in cellular excitability. IMPLICATIONS: Using the patch clamp technique, we showed that T-type calcium channels, which promote cellular excitability, are inhibited by volatile anesthetics in neuronal and neuroendocrine cells, but not in ventricular myocytes. Inhibition of neuronal T-type channels may contribute to the mechanism of action of volatile anesthetics.