Vitamin D receptor activation by paricalcitol and insulin resistance in CKD.

BACKGROUND AND AIMS: The nature of the link (causal vs non-causal) between low 1,25-OH vitamin D and insulin resistance (IR) in patients with chronic kidney disease (CKD) remains elusive. We have now made a post hoc analysis of the effect of vitamin D receptor activation by paricalcitol on IR in the complete dataset of a double-blind, randomized, placebo controlled trial, the Paricalcitol and ENdothelial fuNction in chronic kidneY disease (PENNY). METHODS AND RESULTS: Eighty-eight patients with stage 3-4 CKD were randomized (1:1) to receive 2 µg/day paricalcitol or matching placebo for 12 weeks. IR was measured by five IR indices: the homeostasis model assessment of insulin resistance (HOMA-IR), the quantitative insulin sensitivity check index (QUICKI), the McAuley index, the HOMA corrected for adiponectin (HOMA-AD) and the Leptin-adiponectin ratio (LAR). As compared to placebo, paricalcitol produced the expected small rise in serum calcium (+0.07 mmol/L, P = 0.01) and phosphate (+0.08 mmol/L, P = 0.034) and the expected parathyroid hormone suppression (-96 pg/ml, P < 0.001). However, the drug largely failed to affect the five indices of IR which remained unchanged both in the active and the placebo arm (paricalcitol vs placebo, P ranging from 0.25 to 0.62) and no effect modification of paricalcitol on IR by vitamin D or other parameters was registered. CONCLUSION: Paricalcitol treatment for 12 weeks does not improve IR in patients with stage 3-4 CKD. Low vitamin D receptor activation is not a causal factor for IR in the CKD population.

Serum phosphate modifies the vascular response to vitamin D receptor activation in chronic kidney disease (CKD) patients.

BACKGROUND AND AIMS: Vitamin D receptor activation (VDRA) ameliorates endothelial dysfunction in CKD patients but also increases phosphate and FGF-23, which may attenuate the beneficial effect of VDRA on endothelial function. METHODS AND RESULTS: This is a pre-specified secondary analysis of the PENNY trial (NCT01680198) testing the effect of phosphate and FGF-23 on the flow mediated vasodilatory (FMD) response to paricalcitol (PCT, 2 µg/day) and placebo over a 12-weeks treatment period. Eighty-eight stage G3-4 CKD patients were randomized to PCT (n = 44) and Placebo (n = 44). Endothelial function was assessed by measuring endothelium dependent forearm blood flow (FBF) response to ischemia. The FMD response was by the 61% higher in PCT treated patients than in those on placebo (P = 0.01). Phosphate (+11%, P = 0.039), calcium (+3%, P = 0.01) and, particularly so, FGF23 (+164%, P < 0.001) increased in PCT treated patients. Changes in FMD by PCT associated inversely with phosphate (r = -0.37, P = 0.01) but were independent of FGF-23, calcium and PTH changes. The response to PCT was maximal in patients with no changes in phosphate (1st tertile), attenuated in those with mild-to-moderate rise in phosphate (2nd tertile) and abolished in those with the most pronounced phosphate increase (3rd tertile) (effect modification P = 0.009). No effect modification by FGF-23 and other variables was observed. CONCLUSIONS: The beneficial effect of PCT on endothelial function in CKD is maximal in patients with no or minimal changes in phosphate and it is abolished in patients with a pronounced phosphate rise. These findings generate the hypothesis that the endothelium protective effect by VDRA may be potentiated by phosphate lowering interventions.

[A "lump" in the throat]. / Un "nodo" in gola.

This is a clinical case of a patient suspected to have a lymphoma. Later on, other diagnostic procedures and particularly a neck ultrasound, revealed an idiopathic hyperparathyroidism.

Effect of cholesterol on membrane microheterogeneity: a study using 1,6-diphenyl-1,3,5-hexatriene fluorescence lifetime distributions.

The effect of cholesterol on microheterogeneity of liposomes obtained from saturated and unsaturated phospholipids was studied by measuring the fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH). Data obtained by frequency domain fluorometry have been analyzed either by discrete exponential or continuous lifetime distribution approaches. In egg phosphatidylcholine liposomes, the addition of cholesterol increases the lifetime value or the centre of the lifetime distribution. At high cholesterol concentration, good fits are obtained using a monomodal distribution analysis or single exponential component. At low cholesterol concentration an additional short component of low fractional intensity must be included to obtain a good fit. In dipalmitoylphosphatidylcholine, the addition of cholesterol decreases the long lifetime component centre value both in the gel and in the liquid-crystalline state. The DPH lifetime value is sensitive to the dielectric constant of the probe microenvironment, and cholesterol has been shown to modify water penetration in the bilayer. Using this information our data indicate that cholesterol affects the polarity of the microenvironment in liposomes of unsaturated phosphatidylcholine and saturated phosphatidylcholine in different ways. Although the major conclusions of this paper are obtained using changes of the distribution centre upon cholesterol addition, there are also preliminary indications that the lifetime distribution width decreases as cholesterol is added. We have interpreted this observation as being due to the homogenizing effect of cholesterol.

Fluorescence lifetime distributions of 1,6-diphenyl-1,3,5-hexatriene reveal the effect of cholesterol on the microheterogeneity of erythrocyte membrane.

The fluorescence decay of 1,6 diphenyl-1,3,5-hexatriene (DPH) has been used to characterize aspects of the erythrocyte membrane structure related to the microheterogeneity of the lipid bilayer. The DPH decay has been studied using frequency domain fluorometry and the data analyzed either by a model of discrete exponential components or a model that assumes a continuous distribution of lifetime values. The main intensity fraction was associated with a lifetime value centered at about 11 ns in the erythrocyte membrane, but a short component of very low fractional intensity had to be considered to obtain a good fit to the data. The lifetime value of the long component was insensitive to temperature, while the width of the distribution decreased with increasing temperature. In multilamellar liposomes prepared from phospholipids extracted from the erythrocytes, the long lifetime component showed a temperature dependence. The depletion of 27% of the cholesterol in the erythrocyte membrane induced a broadening of the distribution, suggesting a homogenizing effect of cholesterol. This effect has also been detected in egg phosphatidylcholine at a very low cholesterol/phospholipid molar ratio. The role of cholesterol on membrane heterogeneity is discussed in relation to the effect of cholesterol on water penetration.

N-dansyl-S-nitrosohomocysteine a fluorescent probe for intracellular thiols and S-nitrosothiols.

The fluorescence emission spectrum of N-dansyl-S-nitrosohomocysteine was enhanced approximately 8-fold upon removal of the NO group either by photolysis or by transnitrosation with free thiols like glutathione. The fluorescence enhancement was reversible in that it could be quenched in the presence of excess S-nitrosoglutathione. Attempts were then made to utilize N-dansyl-S-nitrosohomocysteine as an intracellular probe of thiols/S-nitrosothiols. Fluorescence microscopy of fibroblasts in culture indicated that intracellular N-dansyl-S-nitrosohomocysteine levels reached a maximum within 5 min. N-Dansyl-S-nitrosohomocysteine fluorescence was directly proportional to intracellular GSH levels, directly determined with HPLC. N-Dansyl-S-nitrosohomocysteine preloaded cells were also sensitive to S-nitrosoglutathione uptake as the intracellular fluorescence decreased as a function of time upon exposure to extracellular S-nitrosoglutathione.

Membrane heterogeneity in isolated rat hepatocytes and liver plasma membrane subfractions: a comparative study using DPH and its cationic derivative TMA-DPH.

The fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH) and of 1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) has been studied in hepatocytes isolated from rat liver and in isolated plasma membrane subfractions (cLPM, canalicular membranes and bLPM, basolateral membranes) using frequency domain fluorometry. The decay has been analyzed either by using a model of discrete exponential components or a model that assumes a continuous distribution of lifetime values in order to study different aspects of membrane heterogeneity. The results obtained by the two analyses are practically superimposable but the distributional approach allows an evaluation of membrane heterogeneity through the width of the distribution that has shown particularly significant differences when freshly hepatocytes are compared with in vitro aged hepatocytes. Moreover, the comparison of the distributional analysis of the two probes has shown in cLPM a tendency to higher values of the main lifetime component and a narrower distribution width with respect to bLPM. These results indicate changes of membrane domain organization that have been discussed in relation with the specific lipid composition that characterizes the two membrane subfractions. Our results indicate that frequency domain fluorometry may be used to study membrane heterogeneity in intact cells and isolated membranes.

Time-resolved fluorescence of S-100a protein in the absence and presence of calcium and phospholipids.

We have used phase-modulation fluorescence lifetime measurements to study the single Trp residue of the Ca(2+)-binding protein S-100a. Trp fluorescence decay was not exponential for the protein irrespective of the absence or presence of Ca2+. Fluorescence decay was best described by Lorentzian lifetime distributions centered around two components (approx. 3 and 0.7 ns) for protein in absence of Ca2+ and one component (approx. 2.9 ns) for the protein in presence of 2 mM Ca2+. Similar studies were performed with S-100a interacting with cardiolipin, phosphatidylserine or egg phosphatidylcholine, both in absence and in presence of 2 mM Ca2+. Our data suggest that the conformation of the protein and its Ca(2+)-binding properties vary depending on the characteristics of charge and structure of phospholipids.

Plasma membrane changes associated with rat liver regeneration.

The lipid composition and fluidity of plasma membranes have been studied at different stages of liver regeneration (4, 15 and 24 h after surgery). The phospholipid and fatty acid composition is not modified, whereas the cholesterol/phospholipid ratio is lower with respect to control membranes. The modification of the physical properties of the membranes has been studied directly by EPR analysis and indirectly by temperature dependence and cooperativity of some membrane-bound enzymes (Mg2+-ATPase, (Na+ + K+)-ATPase and 5'nucleotidase). Surgical operation or anaesthesia alone causes an early increase in fluidity; such an effect appears to be markedly reduced at a later stage. There seems to be a marked effect of regeneration on plasma membrane fluidity 15 h after partial hepatectomy when several parameters--surface fluidity, cholesterol/phospholipid ratio, and 5'-nucleotidase activity in the presence of concanavalin A -- are modified and indicate an increase in membrane fluidity. It is suggested that this modification of membrane properties could be related to the proliferative process.

Altered platelet membrane dynamic properties in type 1 diabetes.

A modified platelet response to aggregating stimuli is supposed to play a role in the pathogenesis of diabetic macroangiopathy. We studied the fluidity and microheterogeneity of the external surface of the platelet membrane and the activities of the plasma membrane Na+-K+-ATPase and Ca2+-ATPase in 21 men with type 1 diabetes and in 20 control subjects before and after in vitro thrombin addition. In the resting state, platelets from type 1 diabetic patients showed an increased fluidity and microheterogeneity of the platelet membrane, a higher Ca2+-ATPase activity, and a reduced Na+-K+-ATPase activity in comparison with platelets from healthy subjects. The fatty acid composition was also modified, with increased C 16:1 and decreased C 18:0 content. Control cells incubated with thrombin showed a modification of the membrane parameters opposite to the response observed in type 1 cells after the stimulation. The incubation of control platelets in the resting state with high concentrations of glucose modified the fluidity of the plasma membrane Na+-K+-ATPase and Ca2+-ATPase activities in an opposite way in comparison with the alterations observed in type 1 platelets. This study suggests that in type 1 diabetic patients, the platelet membrane responds to activation with a molecular remodeling different from the response of healthy subjects. The abnormal organization of the membrane might contribute to the altered platelet functions in type 1 diabetic patients, but acute exposure to high glucose levels does not seem able to modify the platelet membrane in the way observed in type 1 diabetes.

Reduced Na(+)-K(+)-ATPase activity and plasma lysophosphatidylcholine concentrations in diabetic patients.

A fraction from normal human plasma inhibiting Na(+)-K(+)-ATPase has been recently identified as lysophosphatidylcholine (LPC). The aim of this study was to investigate the existence of a relationship between the activity of the cellular membrane Na(+)-K(+)-ATPase and plasma LPC in human diabetes. We studied 10 patients with insulin-dependent-diabetes mellitus (IDDM), 14 patients with non-insulin-dependent diabetes mellitus (NIDDM), and 10 sex- and age-matched control subjects. Plasma LPC concentrations were increased in both IDDM and NIDDM patients compared with control subjects. Na(+)-K(+)-ATPase activity was reduced in both groups of patients in erythrocyte and platelet membranes. There was a significant correlation between the concentrations of plasma LPC and Na(+)-K(+)-ATPase activity in both erythrocyte and platelet membranes (P < 0.01). To investigate the effect of LPC on the enzyme, Na(+)-K(+)-ATPase activity was determined in erythrocyte membranes obtained from six healthy subjects after in vitro incubation with increasing concentrations of LPC (1-10 microM). Enzymatic activity was significantly reduced by in vitro LPC at a concentration of 2.5 microM, with a further decrease at 5 microM. These data suggest that the decrease in Na(+)-K(+)-ATPase activity in diabetes might be due to increased LPC concentrations.

Interaction of S-100b protein with cardiolipin vesicles as monitored by electron spin resonance, pyrene fluorescence and circular dichroism.

The interaction of S-100b protein with cardiolipin (CL) vesicles has been studied by electron spin resonance, pyrene fluorescence, and circular dichroism. Electron spin resonance and pyrene fluorescence data indicate that S-100b binds to the polar surface of vesicles Ca2+-independently. In the presence of Ca2+, S-100b potentiates the Ca2+-induced clustering of the polar headgroups of CL molecules and causes a further reduction in the Ca2+-dependent decrease in the lateral mobility of the pyrene inserted into the lipid bilayer, which points to an effect of the protein on the hydrophobic core of the lipid bilayer through a larger perturbation of its polar surface. Circular dichroism analyses indicate that CL vesicles cause a decrease in the alpha-helical content of S-100b, analogous to that produced by Ca2+ and that the effects of CL vesicles and of Ca2+ on the secondary structure of the protein are supra-additive. By this technique, we found that the affinity of Ca2+ for S-100b increases substantially in the presence of CL vesicles, even in the presence of physiologic concentrations of KCl, suggesting that once S-100b had interacted with CL vesicles it assumes a new conformation in which its Ca2+-binding properties are greatly enhanced. These results are discussed in relation to binding of S-100b proteins to natural membranes, and to a possible involvement of S-100b in the regulation of membrane structural organization.

S-100b protein regulates aggregation and fusion of cardiolipin vesicles.

We have recently shown that S-100b protein interacts with the polar surface of cardiolipin vesicles [6]. This interaction produces changes in the secondary structure of S-100b as well as changes in the structural organization of cardiolipin vesicles. We report here on the effects of S-100b on cardiolipin vesicles as investigated by turbidity, terbium-dipicolinate fluorescence and freeze-fracture. Experiments were carried out in the absence and in the presence of Ca2+. In the absence of Ca2+ (0.1 mM EDTA), S-100b favors the aggregation and fusion of vesicles to some extent. Under these conditions, electron microscope analyses reveal the presence of fused vesicles along with particles similar to those observed in protein reconstituted systems or to lipid particles observed during fusional processes. In the presence of Ca2+, S-100b counteracts the Ca2(+)-dependent tendency of vesicles to aggregate and fuse. Under these conditions, bilayer phases along with hexagonal phases can be observed by electron microscopy. The latter effects of S-100b are not due to chelation of Ca2+ because of the relative concentrations of S-100b and Ca2+ under our experimental conditions and since much larger concentrations of EDTA are required to produce the S-100b effects. We propose that the dimeric nature of S-100b plays a major role in these events. In the absence of Ca2+, the S-100b molecules probably cross-link adjacent vesicles, one subunit contacting one vesicle and the other subunit contacting another vesicle through electrostatic bonds. In the presence of Ca2+, due to the large changes occurring in the conformation of the protein (which loses about 52% of its alpha-helical content), S-100b associates strongly with the polar surface of individual vesicles, thus generating some kind of physical barrier to aggregation and fusion of vesicles.

Time-resolved fluorescence of S-100a protein: effect of Ca2+, Mg2+ and unilamellar vesicles of egg phosphatidylcholine.

Phase-modulation fluorescence lifetime measurements were used to study the single Trp residue of the Ca(2+)-binding protein S-100a both in the absence and in the presence of Ca2+ and/or Mg2+. Trp fluorescence decay for the protein was satisfactorily described by Lorentzian lifetime distributions centered around two components (approximately 4 ns and 0.5 ns). Lifetime values were unchanged by 2 mM Ca2+, but the fractional intensity associated with longer lifetime increased up to 75%. In the presence of Mg2+, the Ca2+ induced increase of the fractional intensity associated with longer lifetime was only 57%. For the protein in buffer, about the 85% of the recovered anisotropy was associated to a rotational correlation time of 6.7 ns. After the addition of Ca2+, this value was increased to 16.08 ns. In the presence of Mg2+, Ca+2 increased the rotational correlation time to 33.75 ns. Similar studies were performed with S-100a interacting with egg phosphatidylcholine vesicles (SUV). Our data suggest that the conformation of the protein may be influenced by structural features of the lipidic membrane. Moreover, data obtained in the presence of Mg2+ indicate some interaction between lipids and S-100, likely mediated by this ion.

Protective effect of paraoxonase activity in high-density lipoproteins against erythrocyte membranes peroxidation: a comparison between healthy subjects and type 1 diabetic patients.

High-density lipoproteins (HDL) plays a key role in the protection against oxidative damage of lipoprotein and biological membranes. The aim of the present study was to investigate the relationship between the antioxidant role of HDL and the HDL-paraoxonase (PON) activity in healthy subjects and in type 1 diabetic patients. Moreover, the ability of HDL of controls and diabetic patients to protect and/or repair biological membranes from oxidative damage was studied. HDL were isolated from 31 type 1 diabetic patients and 31 sex- and age-matched healthy subjects and immediately used to evaluate lipid hydroperoxides and HDL-PON activity. Erythrocyte membranes obtained from healthy subjects were oxidized with 2,2-azo-bis(2-aminidinopropane)dihydrochloride and then incubated in the presence of HDL isolated from healthy or type 1 diabetic subjects, with measurements of membrane lipid hydroperoxides before and after the incubation. HDL from type 1 diabetic patients showed higher levels of lipid hydroperoxides and a lower activity of HDL-PON than healthy subjects. Moreover, HDL of type 1 diabetic patients protected less efficiently erythrocyte membranes against oxidative damage compared with HDL from healthy subjects. A negative correlation was found between HDL-PON activity and the levels of hydroperoxides of HDL, confirming the relationship between PON and lipid peroxidation and suggesting that subjects with low PON activity are more exposed to oxidative damage than subjects with high PON activity. The ability of HDL to protect erythrocyte membranes was positively correlated with HDL-PON activity and negatively correlated with the levels of lipid hydroperoxides of HDL of healthy subjects. These results confirm a linkage between PON activity and lipid peroxidation of lipoproteins and suggest that the ability of HDL to protect erythrocyte membranes might be related to the PON activity. It might be hypothesized that the decrease of PON activity in diabetic patients and the lower HDL protective action against membrane peroxidation could contribute to acceleration of arteriosclerosis in type 1 diabetes mellitus.

Modification induced by homocysteine and low-density lipoprotein on human aortic endothelial cells: an in vitro study.

The aim of the present study was to investigate the effect exerted by low-density lipoprotein (LDL) modified by homocysteine (Hcy)-thiolactone (Hcy-LDL) on functional properties on human endothelial cells. Hcy-thiolactone, a reactive product formed in human cells from enzymatic conversion of Hcy, was hypothesized to play an important role in Hcy-induced vascular damages. Using endothelial cultured cells [human aortic endothelial cells (HAEC)] as cellular model, we evaluated nitric oxide (NO) production, cytoplasmic Ca(2+) levels, Na(+)/K(+)-ATPase activity, and peroxynitrite production in cells incubated in the presence of control LDL or Hcy-LDL. Homocysteinylation of LDL was carried out by incubation of LDL, isolated from plasma of healthy subjects, with 100 microm Hcy-thiolactone. A significant increase in cytoplasmic Ca(2+) levels and peroxynitrite production and a decrease in Na(+)/K(+)-ATPase and NO production in HAEC incubated with Hcy-LDL compared with HAEC incubated with control LDL were observed. Moreover, a positive correlation was found between Na(+)/K(+)-ATPase activity and cytoplasmic Ca(2+) content and between peroxynitrite activity and cytoplasmic Ca(2+) content. In conclusion, our results demonstrated that LDL homocysteinylated in vitro induced alterations of functional properties and NO metabolism of human endothelial cells.

Glycated low density lipoproteins modify platelet properties: a compositional and functional study.

The interaction between low density lipoproteins (LDL) and platelets might play a central role in the development of atherosclerosis in diabetes. The aim of the present study was to investigate whether the glycation of LDL is associated with modifications of their physico-chemical and functional properties and to study the action of glycated LDL (glycLDL) on platelets. LDL and platelets were isolated from 15 healthy subjects. The content of thiobarbituric acid-reactive substances and the generalized polarization of the fluorescent probe Laurdan were determined in LDL glycated in vitro. Platelets were incubated with native LDL, GlycLDL, and minimally oxidized LDL, and the following parameters were evaluated: platelet aggregation, nitric oxide production, intracellular Ca(2+) concentrations, Na(+)/K(+)-adenosine triphosphatase (Na(+)/K(+)-ATPase), and Ca(2+)-ATPase activities. GlycLDL showed increased thiobarbituric acid-reactive substance levels, a red shift of the Laurdan emission maximum, and a decrease in generalized polarization, indicating a higher polarity and a reduced molecular order compared with native LDL. GlycLDL caused a significant increase in platelet nitric oxide production, intracellular Ca(2+) concentration, and aggregating response to ADP; an inhibition of the platelet membrane Na(+)/K(+)-ATPase activity; and a stimulation of Ca(2+)-ATPase activity. Minimally oxidized LDL did not cause statistically significant changes in the parameters studied. The present work demonstrates that glycation induces compositional and structural changes in LDL and suggests that an altered interaction between glycLDL and platelets might play a role in the vascular complications of diabetes.

Influence of low density lipoprotein from insulin-dependent diabetic patients on platelet functions.

In the present work we studied in vitro the action of low density lipoproteins (LDL) isolated from normolipemic insulin-dependent diabetic (IDDM) patients on transmembrane cation transport, nitric oxide synthase (NOS) activity, and aggregating response to stimuli of platelets from healthy subjects to elucidate whether the modified interaction between circulating lipoproteins and cells might be one of the pathogenetic mechanisms of the increased platelet activation in IDDM. LDL were obtained by discontinuous gradient ultracentrifugation from 15 IDDM out-patients and 15 sex- and age-matched healthy subjects and used for incubation experiments with control platelets. Lipid composition and hydroperoxide concentrations were studied in LDL. Platelet aggregation responses to ADP, NOS activity, cytosolic Ca2+ concentrations, and platelet membrane Na+/K+-adenosine triphosphatase (Na+/K+-ATPase) and Ca2+-ATPase activities were measured after incubation. IDDM LDL showed an increased lysophosphatidylcholine content compared with that of control LDL. IDDM LDL significantly increased the platelet aggregating response to ADP, cytosolic Ca2+ concentrations, and plasma membrane Ca2+-ATPase activity and significantly reduced NOS activity and platelet membrane Na+/K+-ATPase activity compared with those of platelets incubated in buffer or cells incubated with control LDL. The effects exerted by IDDM LDL on platelet suspensions from healthy subjects mimic the alterations observed in platelets from diabetic subjects in basal conditions. Both the decreased activity of NOS and the higher cytoplasmic concentrations of Ca2+ might cause increased platelet activation, as observed in IDDM. In conclusion, the present study suggests a new mechanism with a potential role in the early development of atherosclerosis in diabetic patients, i.e. an altered interaction between circulating lipoproteins and platelets.

Changes of fluorescence anisotropy in plasma membrane of human polymorphonuclear leukocytes during the respiratory burst phenomenon.

Steady state fluorescence anisotropy (rs) of TMA-DPH was measured to study the effect of respiratory burst activation with PMA, FMLP, and PAF on the physico-chemical structure of PMNs plasma membrane. Our results show a significant increase in rs during the respiratory burst activation. In the presence of NADPH-oxidase inhibitor DPI, only PAF induces changes in rs values. This suggests a non-specific effect of PAF on plasma membrane. Azide, which induces a supranormal release of H2O2, fails to increase the basal rs value after activation. Moreover, the catalase does not abolish the increase in rs induced upon activation. This rules out the possibility that changes of rs during the respiratory burst activation are attributed mainly to H2O2 release. We conclude that multiple processes accompanying the respiratory burst activation are responsible for the changes in the physico-chemical properties of PMNs plasma membrane.

The transmembrane gradient of the dielectric constant influences the DPH lifetime distribution.

The fluorescence lifetime distribution of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) in egg-phosphatidylcholine liposomes was measured in normal and heavy water. The lower dielectric constant (by approximately 12%) of heavy water compared with normal water was employed to provide direct evidence that the drop of the dielectric constant along the membrane normal shifts the centers of the distribution of both DPH and TMA-DPH to higher values and sharpens the widths of the distribution. The profile of the dielectric constant along the membrane normal was not found to be a linear gradient (in contrast to [1]) but a more complex function. Presence of cholesterol in liposomes further shifted the center of the distributions to higher value and sharpened them. In addition, it resulted in a more gradient-like profile of the dielectric constant (i.e. linearization) along the normal of the membrane. The effect of the change of dielectric constant on the membrane proteins is discussed.