Vitamin D and adenosine triphosphatase dependent on divalent cations in rat intestinal mucosa.

Intestinal brush borders prepared from vitamin D-deficient rats demonstrate increased susceptibility in vitro to fragmentation by shear forces or to loss of microvillus enzymes on treatment with EDTA. These effects are relatively nonspecific and are also observed in normal rats starved for 48 h. They may underlie prior observations that purport to demonstrate a vitamin D-dependent increase in brush border Ca-dependent ATPase. In addition, however, vitamin D increases ATPase activity dependent on certain divalent cations, including Ca and Zn, in whole-particulate suspensions pelleted by high-speed centrifugation of mucosal homogenates. This action is independent of changes in other microvillus enzymes, i.e. disaccharidases, and tissue distribution and cation specificity studies support the hypothesis that the mucosal whole-particulate ATPase is related to transport of Ca, Zn, and possibly other divalent cations.

Fluorescence polarization studies of rat intestinal microvillus membranes.

Rat intestinal microvillus membranes and lipid extracts prepared from them have been studied by fluorescence polarization with three lipid-soluble fluorophores: diphenylhexatriene, retinol, and anthroyl-stearate. The degree of fluorescence polarization of diphenylhexatriene, which provides an index of the "microviscosity" of the lipid regions of the membrane, is exceptionally high in microvillus membranes, the highest yet reported in normal biological membranes. Both the membrane proteins and lipids were found to contribute to the high values. With each of the three probes the polarization values are higher in ileal microvillus membranes as compared to membranes from proximal intestinal segments. Temperature-dependence studies of the fluorescence polarization of diphenylhexatriene and anthroylstearate demonstrate a phase transition in microvillus membranes and in liposomes prepared from their lipid extracts at approximately 26+/-2 degrees C. Ambient pH influences markedly the diphenylhexatriene fluorescence polarization in microvillus membranes but has little effect on that of human erythrocyte ghost membranes. The "microviscosity" of jejunal microvillus membranes is maximal at pH 6.5-7.0 and decreases as much as 50% at pH 3.0, an effect which depends largely upon the membrane proteins. Addition of calcium ions to suspensions of microvillus membranes increases the fluorescence polarization of retinol and anthroyl-stearate, but not that of diphenyl-hexatriene. This confirms the localization of the last compound to the hydrophobic interior of the membrane, relatively distant from the hydrophilic head groups of the polar lipids. Microvillus membrane proteins solubilized with Triton X-100 give relatively high fluorescence polarization and intensity values with retinol, suggesting the presence of binding proteins which could play a role in the normal absorptive mechanism for the vitamin.

Transport of monosaccharides. I. Asymmetry in the human erythrocyte mechanism.

Transport of D-glucose across human erythrocyte membranes occurs via a facilitated diffusion process which demonstrates influx-efflux asymmetry. The mechanism of the asymmetry has been studied by estimating unidirectional fluxes in the presence or absence of trans equilibrium hexose. In the absence of transhexose, the half-saturation constant for efflux at 15 degrees C was approximately 10 mM as compared with 27 mM for influx; the corresponding values for maximal transfer rates (mumol/min per ml cell H(2)O) were approximately 51 vs. 18. The estimation of kinetic parameters, including the constant F(s), which is the ratio of maximal transfer rate/half-saturation constant, indicates a unique effect of intracellular hexose on the transfer system. Further evidence to support this conclusion was obtained by studying the effects of noncompetitive inhibitors on efflux vs. influx. N-ethylmaleimide, p-chloromercuribenzenesulfonate, and dichloroallyldiethylstilbestrol all inhibited efflux much more than influx. Glucose rendered the transport system more reactive to N-ethylmaleimide as assayed by efflux, whereas influx was much less affected. The results support the hypothesis that the transport system exists in two states. Transition from one state to the other is dependent on the presence of intracellular hexose.

Membrane lipid dynamics in human promyelocytic leukemia cells sensitive and resistant to 12-O-tetradecanoylphorbol-13-acetate induction of differentiation.

A series of fluorescent probes was used to analyze membrane lipid dynamics in promyelocytic leukemic cells sensitive (HL-60) or resistant (R-55) to phorbol diester induction of cell differentiation. When examined with the probe 1,6-diphenyl-1,3,5-hexatriene, which can penetrate the plasma membrane and intercalate in the lipids of both leaflets of the plasma membrane, as well as in organellar membranes, R-55 cells were found to have higher fluorescence anisotropy values, indicative of decreased lipid fluidity, as compared to HL-60 cells. In contrast, when HL-60 and R-55 cells were compared using a series of membrane-impermeant fluorophores (stachyose derivatives of anthroyloxystearate and pyrenebutyryl hydrazide) that incorporate only into the outer hemileaflet of the plasma membrane, no difference was observed in membrane lipid fluidity. Exposure to 12-O-tetradecanoylphorbol-13-acetate (10 ng/ml) for 24 hr decreased the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene in both HL-60 and R-55 cells, whereas by 48 hr only the HL-60 cells displayed the reduction. No effect on the fluorescence anisotropy of 1-(4'-trimethylammonium phenyl)-6-phenyl-1,3,5-hexatriene, which is believed to localize in the plasma membrane, was observed in R-55 cells exposed to 12-O-tetradecanoylphorbol-13-acetate (10 or 100 ng/ml), whereas HL-60 cells treated with 12-O-tetradecanoylphorbol-13-acetate (10 ng/ml) showed a marked reduction in the fluorescence anisotropy. These observations suggest that the ability of HL-60 cells to respond to 12-O-tetradecanoylphorbol-13-acetate may be affected by the physical state of the plasma membrane lipids and that the resistant phenotype is associated with decreased fluidity of either the inner leaflet of the plasma membrane and/or of the cytosolic organellar membranes.

A dietary regimen alters hepatocyte plasma membrane lipid fluidity and ameliorates ethinyl estradiol cholestasis in the rat.

A dietary regimen which induces hepatic fatty acyl desaturase activities increases the lipid fluidity of hepatocyte plasma membranes, both in normal rats and in animals treated with ethinyl estradiol to produce cholestasis. In the cholestatic animals the fluidity change is accompanied by a significant increase in bile flow rate of approx. 33%.

Calcium alters the acyl chain composition and lipid fluidity of rat hepatocyte plasma membranes in vitro.

Calcium ion decreases the lipid fluidity of isolated rat hepatocyte plasma membranes by modulating the activity of membrane enzymes which alter the lipid composition. To explore the mechanism of the effect of the cation, eight fluorophores were used to assess lipid fluidity via estimations of either steady-state fluorescence polarization or excimer fluorescence intensity. The results demonstrate that the reduction in fluidity occurs in the hydrophobic interior of the bilayer and that both the dynamic and static (lipid order) components of fluidity are affected by treatment with calcium. Analysis of the membrane lipids demonstrates that calcium treatment decreases the arachidonic acid content of the polar lipid fraction and, thereby, reduces the double-bond index of the fatty acids. This change in composition, which is expected to reduce the lipid fluidity, may result from activation by calcium of the endogenous hepatocyte plasma membrane phospholipase A2.

Lipid composition and fluidity of rat enterocyte basolateral membranes. Regional differences.

The lipid composition and fluidity of basolateral membranes prepared from the mucosa of the proximal, middle and distal thirds of the rat small intestine were determined. Fluidity, as assessed by the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene and a series of anthroyloxy fatty acid derivatives, is decreased in the distal third as compared to the proximal segments. This pattern is similar to that described previously for microvillus membranes. The decrease in fluidity of the distal as compared to the proximal membranes results from an increase in cholesterol content, cholesterol/phospholipid molar ratio and degree of saturation of the fatty acid residues. In the middle and distal thirds of the gut, the degree of saturation of the fatty acid residues is higher in microvillus as compared to basolateral membranes, accounting in part for the characteristically lower fluidity of the luminal membranes. The specific activity of the basolateral membrane (Na+ + K+)-dependent adenosine triphosphatase is significantly lower in the distal as compared to the proximal and middle thirds of the intestinal mucosa. Studies of the binding of [3H]ouabain indicate that this pattern results from fewer enzyme sites in the distal membranes.

Membrane lipids can modulate guanylate cyclase activity of rat intestinal microvillus membranes.

Studies of the temperature dependence (10-40 degrees C) of guanylate cyclase in rat intestinal microbillus membranes reveal a change in energy of activation (slope of the Arrhenius plot) at 30 +/- 1 degree C. The break point temperature corresponds to the lipid thermotropic transition in these membranes previously characterized by differential scanning calorimetry (range: 23-39 degrees C; peak temperature, 31 degrees C). The break point temperature for guanylate cyclase also corresponds to that of a number of other microbillus membrane enzymes and of D-glucose transport. These activities are defined as "intrinsic" membrane activities by this operational criterion. Treatment with the nonionic detergent Lubrol WX increased the guanylate cyclase activity 4- to 8-fold and removed the discontinuity in the Arrhenius plot.

Variations in dietary triacylglycerol saturation alter the lipid composition and fluidity of rat intestinal plasma membranes.

Rats were maintained on nutritionally complete diets enriched in unsaturated (corn oil) or saturated (butter fat) triacylglycerols. After 6 weeks, significant differences in the lipid composition and fluidity of a number of intestinal membranes were observed. The corn oil diet (enriched mainly in linoleic acid) increased the overall unsaturation of the acyl chains and enhanced the lipid fluidity, as assessed by the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene, of enterocyte microvillus and basolateral membranes and of colonocyte basolateral membranes. Concomitantly, the cholesterol content and the cholesterol/phospholipid molar ratio were increased in the microvillus but not in the basolateral membranes. The increased cholesterol in ileal microvillus membranes can result from enhanced cellular biosynthesis, since ileal slices from rats fed the unsaturated diet incorporated [14C]octanoate more rapidly into digitonin-precipitable sterol. Increased fluidity of the enterocyte microvillus and basolateral membranes, respectively, enhanced the enzyme specific activities of p-nitrophenylphosphatase and (Na+ + K+)-dependent adenosine triphosphatase. The results indicate that the lipid composition, fluidity and enzyme activities of intestinal plasma membranes can be altered by dietary means. Moreover, rat enterocytes possess regulatory mechanisms which modulate the cholesterol content of the microvillus membranes so as to mitigate changes in lipid fluidity.

Lipid fluidity and composition of intestinal microvillus membranes isolated from rats of different ages.

The lipid composition and fluidity of microvillus (luminal) membranes isolated from the small intestines of Fisher 344 rats aged 6, 17, and 117 weeks were compared. Lipid fluidity, as assessed by the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene, was significantly greater in rats aged 6 weeks as compared to 17 or 117 weeks. A lipid thermotropic transition was observed at 17.5 +/- 1.3 degrees C in the membranes of the youngest group, approx. 5-6 degrees C lower than that of the older animals. The differences in lipid composition which account for the higher fluidity of the youngest preparations include a decreased cholesterol/phospholipid molar ratio in both the proximal and distal halves of the small intestine and, in the proximal half alone, increases in the lipid/protein ratio and double bond index. The foregoing reduction in cholesterol/phospholipid ratio derives mainly from a higher content of total phospholipid, and the increment in double bond index results from an increase in arachidonic acid residues. The results demonstrate an age-dependent decrease in fluidity of intestinal microvillus membranes in the early post-weaning period in the rat. This pattern was unlike that of the microvillus membrane p-nitrophenylphosphatase, whose specific activity declined progressively in the older age groups.

Pressure dependence of pyrene excimer fluorescence in human erythrocyte membranes.

The intensity of pyrene excimer fluorescence in human erythrocyte membranes and in sonicated dispersions of the membrane lipid (liposomes) was examined as a function of pressure (1-2080 bar) and temperature (5-40 degrees C). Higher pressure or lower temperature decreased the excimer/monomer intensity ratios. A thermotropic transition was detected in both membranes and liposomes by plots of the logarithm of the excimer/monomer intensity ratio versus 1/K. The transition temperature of the membranes was 19-21 degrees C at 1 bar and 28-31 degrees C at 450 bar, a shift with pressure of approx. 20-22 K per kbar. Corresponding transition temperatures of the liposomes were 21 degrees C at 1 bar and 33 degrees C at 450 bar, a shift of approx. 27 K per kbar. The observed pressure dependence of the thermotropic transition temperature is similar to that reported for phospholipid bilayers and greatly exceeds that of protein conformation changes. In concert with the liposome studies the results provide direct evidence for a lipid transition in the erythrocyte membrane.

Effects of benzyl alcohol on erythrocyte shape, membrane hemileaflet fluidity and membrane viscoelasticity.

The effects of benzyl alcohol on cell shape, hemileaflet lipid fluidity and membrane rheology of human red blood cells were studied. Membrane fluidity was assessed by determining the fluorescence anisotropy of permeant probes (1,6-diphenyl-1,3,5-hexatriene,12-(9-anthroyloxy)stearate, 2-(9-anthroyloxy)stearate) and a new impermeant probe (N-stachyosylsuccinic acid dihydrazide-2-(9-anthroyloxy)stearate). Measurements made on intact red blood cells reflected primarily the outer leaflet fluidity while measurements made on red blood cells ghosts reflected the fluidity of both leaflets. Membrane viscoelasticity was determined by micropipette aspiration. Treatment of intact red blood cells with benzyl alcohol up to 50 mM caused progressive stomatocytic shape change but no change in membrane viscoelasticity, 1,6-diphenyl-1,3,5-hexatriene anisotropy or stachyosyldihydrazide-2(9-anthroyloxy)stearate correlation time; similar treatment of leaky ghosts yielded decreases in 1,6-diphenyl-1,3,5-hexatriene anisotropy and stachyosyldihydrazide-2(9-anthroyloxy)stearate correlation time. With benzyl alcohol above 50-60 mM, intact red blood cells became echinocytic, and decreases in 1,6-diphenyl-1,3,5-hexatriene anisotropy and stachyosyldihydrazide-2(9-anthroyloxy)stearate correlation time occurred in both intact cells and ghosts; there was no change in membrane viscoelasticity. These results indicate that benzyl alcohol up to 50 mM affects primarily the inner leaflet of the red blood cell membrane and that higher concentrations affect both leaflets. These increases in membrane fluidity are not associated with changes in membrane viscoelasticity. This study illustrates the use of fluorescence techniques to monitor specifically the lipid fluidity of each hemileaflet of the erythrocyte membrane.

Lipid fluidity of hepatocyte plasma membrane subfractions and their differential regulation by calcium.

Rat hepatocyte plasma membranes were subfractionated by several methods into canalicular, sinusoidal and mixed contiguous plus sinusoidal membranes. Assessment of lipid fluidity by steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene and 12-(9-anthroyloxy)stearate indicates that the canalicular fraction is less fluid than the other membranes. Incubation with calcium decreases the fluidity of the sinusoidal and contiguous membranes by altering the lipid composition, an action which is not reversed by subsequent chelation of the cation. This effect of calcium is not observed in canalicular membranes.

Increased rigidity of red blood cell membrane in young spontaneously hypertensive rats.

The micropipette test was used to study the effects of age on the elasticity of red blood cell (RBC) membrane in spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar-Kyoto rats (WKY), ranging from 3 to 23 weeks of age. The development of hypertension in the SHR started at 3 weeks and was fully established at 7 to 8 weeks. In the developmental phase of hypertension (3-5 weeks), the SHR showed a significant increase in RBC membrane elastic modulus (i.e., a decrease in RBC membrane deformability) when compared with the age-matched normotensive control rats (WKY). After the establishment of hypertension (7-8 weeks), however, the deformability of the RBC membrane of SHR improved and became comparable to that of the WKY. These results indicate that abnormal erythrocyte membrane elasticity is an early event in SHR and that adaptive recovery occurs when hypertension is fully developed.

Inheritance of salt-dependent hypertension in the inbred Dahl rat.

The mode of inheritance of salt-dependent hypertension in the inbred Dahl salt-sensitive rat strain was examined by genetic crosses with the corresponding salt-resistant strain. The blood pressure responses to ingestion of a high NaCl (8%) diet defined three phenotypes: early onset (within 17 days) of systolic hypertension, defined as greater than or equal to 140 mm Hg, the parental salt-sensitive phenotype; late onset of systolic hypertension requiring 50 to 60 days in males and more than 200 days in females, characteristic of the F1 progeny; and normotension, less than 140 mm Hg, the parental salt-resistant phenotype. The frequencies of the phenotypes observed among 91 F2 progeny and 45 progeny of the backcross to parental salt-sensitive animals agree well with values predicted by a model in which two autosomal, unlinked, allelic loci, termed alpha and beta, determine the inheritance. For F2 male progeny, the predicted frequencies of early-onset hypertension, late-onset hypertension, and normotension are 0.1875, 0.5625, and 0.25, respectively, and the corresponding observed frequencies were 0.156, 0.50, and 0.34 (X2 = 0.48, P > .50). F1 progeny of reciprocal parental crosses were maintained on the 8% NaCl diet for 255 days. Male F1 rats developed systolic hypertension sooner than did females. From 60 to 200 days, the average blood pressure value within each group remained approximately stable; the male values exceeded those for females (P < .01); and the direction of the parental cross significantly influenced (P < .05) the levels in males and females, suggestive of genomic imprinting.

Elevation of plasma immunoglobulin A in the spontaneously hypertensive rat.

Prior studies describe deficiencies of T-cell-mediated immunity in the spontaneously hypertensive rat (SHR) strain of Okamoto and Aoki. This report describes an alteration of humoral immunity: elevation of the plasma concentration of immunoglobulin (Ig) A and of circulating IgA autoantibodies to single-stranded DNA, double-stranded DNA, and thyroglobulin. The increased plasma IgA levels are evident in prehypertensive SHR, hence not secondary to the hypertension, and they result mainly from increments in polymeric IgA. Plasma IgA content also varied concordantly with the level of systolic blood pressure as influenced by age (older > younger) and gender (male > female) in both the SHR and control Wistar-Kyoto rat strains. Strain differences in plasma IgG or IgM were not observed. Studies of peripheral blood lymphocytes indicate that increased production of IgA is one mechanism for the increment in plasma content. The number of blood lymphocytes capable of producing IgA in vitro in response to the mitogen lipopolysaccharide is increased in SHR. When cultured in the absence or presence of lipopolysaccharide, peripheral blood lymphocytes of SHR secrete more IgA in vitro than do cells of the control strain. No significant strain differences in biliary or renal excretion of IgA were observed. The observed alterations of IgA in the SHR either are causative factors in the development of the hypertension or are the products of an epiphenomenon in which IgA and blood pressure are affected separately, but in parallel, by causative factors related to rat strain, age, and gender.

Informed consent and tardive dyskinesia.

To determine whether a formalized informing process transmitted knowledge concerning the risks and benefits of neuroleptic medication, particularly the risk of tardive dyskinesia, to stable schizophrenic outpatients, the authors administered a multiple-choice questionnaire to 21 patients who were read a standardized information form and 27 patients who were not. The mean scores for the informed patients were significantly higher, and the differences between the two groups remained significant at 6-month follow-up. The information process had no adverse effects on frequency of psychiatric admission, noncompliance with medication, or the need for increased antipsychotic medication.

Macrophage phagocytosis and membrane fluidity in mice: the effect of age and dietary protein.

Male C57BL/6NNia mice were used to investigate the effects of age and dietary protein intake on Fc and C3b receptor-mediated phagocytosis and on membrane fluidity. Six-month-old (adult) and 24-month-old (aged) mice were fed a 6% or 25% protein diet for 3, 5, or 6 weeks at which time thioglycollate elicited peritoneal macrophages were isolated. Both binding of IgG-coated sheep red blood cells to the macrophages and ingestion via the Fc-receptor were identical in all 4 groups after 3 and 6 weeks of feeding but were decreased at 5 weeks in the aged animals fed 6% protein. Phagocytosis via the C3b receptor was not depressed in either age group fed the low protein diet; it was, however, augmented significantly in the aged animals fed the 25% protein diet for 5 and 6 weeks. Membrane fluidity of the plasma membrane outer hemileaflet was monitored with an impermeant fluorescent probe. No changes were observed between adult and aged mice maintained up to 6 weeks on the diets.

Cytotoxic lymphocyte gene expression in peripheral blood leukocytes correlates with rejecting renal allografts.

BACKGROUND: We have shown previously that heightened expression of the cytotoxic lymphocyte (CL) effector genes perforin (P), granzyme B (GB), and Fas ligand (FasL), is closely correlated with acute allograft rejection, particularly when two or more target genes are up-regulated. METHODS: We used quantitative reverse transcription-polymerase chain reaction to analyze CL gene expression from peripheral blood leukocytes (PBLs) and renal allograft biopsies in 31 paired samples of PBLs and renal tissue from 25 renal allograft recipients. Our aims were (1) to determine whether the expression of CL gene expression in PBLs correlates with expression of these genes in renal allograft biopsy tissue and (2) to determine whether CL gene expression in PBLs correlates with the histological diagnosis. RESULTS: Coordinate gene expression in PBLs and acutely rejecting allografts was found in 9/11 (82%) for P, 07/11 (64%) for GB, and 10/11 (91%) for FasL. Coordinate absence was found in 15/20 (75%) for P, 17/20 (85%) for GB, and 16/20 (80%) for FasL in nonrejecting allografts. Furthermore, up-regulation of any two genes in PBLs correlated with pathological diagnosis of rejection with excellent positive (100%) and negative (95%) predictive values. CONCLUSION: Coordinate CL gene expression in PBLs and the allograft is usually detected. CL gene expression in PBLs is closely associated with a pathologic diagnosis of rejection. CL gene expression in PBLs may serve as a noninvasive method of monitoring for renal allograft rejection.

Acute pulmonary embolism triggered by the act of defecation.

Pulmonary embolism associated with the act of defecation has not been previously well described. Recently, we reported our experience with four patients who presented to us over a 12-month period with syncope, near syncope, or sudden death following the act of defecation. In all four cases, acute pulmonary embolism was shown to be the etiology of the defecation-associated events. A retrospective chart review of all patients with the diagnosis of pulmonary embolism at our institution over a three-year period yielded five additional patients with the diagnosis of defecation-associated pulmonary embolism. These nine patients accounted for 6.8 percent of all patients with a discharge diagnosis of pulmonary embolism seen at our institution during the three-year study period. Six of the nine patients died from their defecation-associated pulmonary embolism. These six deaths accounted for 25 percent of all deaths from pulmonary embolism seen at our institution during the study period. Based on our experience, we suggest that the act of defecation may trigger the development of acute pulmonary embolism in some patients with deep vein thrombosis.