Lipid raft components cholesterol and sphingomyelin increase H+/OH− permeability of phosphatidylcholine membranes

H+/OH− permeation through lipid bilayers occurs at anomalously high rates and the determinants of proton flux through membranes are poorly understood. Since all life depends on proton gradients, it is important to develop a greater understanding of proton leak phenomena. We have used stopped-flow fluorimetry to probe the influence of two lipid raft components, chol (cholesterol) and SM (sphingomyelin), on H+/OH− and water permeability. Increasing the concentrations of both lipids in POPC (palmitoyl-2-oleoyl phosphatidylcholine) liposomes decreased water permeability in a concentration-dependent manner, an effect that correlated with increased lipid order. Surprisingly, proton flux was increased by increasing the concentration of chol and SM. The chol effect was complex with molar concentrations of 17.9, 33 and 45.7% giving 2.8-fold (P<0.01), 2.2-fold (P<0.001) and 5.1-fold (P<0.001) increases in H+/OH− permeability from a baseline of 2.4×10−2 cm/s. SM at 10 mole% effected a 2.8-fold increase (P<0.01), whereas 20 and 30 mole% enhanced permeability by 3.6-fold (P<0.05) and 4.1-fold respectively (P<0.05). Supplementing membranes containing chol with SM did not enhance H+/OH− permeability. Of interest was the finding that chol addition to soya-bean lipids decreased H+/OH− permeability, consistent with an earlier report [Ira and Krishnamoorthy (2001) J. Phys. Chem. B 105, 1484–1488]. We speculate that the presence of proton carriers in crude lipid extracts might contribute to this result. We conclude that (i) chol and SM specifically and independently increase rates of proton permeation in POPC bilayers, (ii) domains enriched in these lipids or domain interfaces may represent regions with high H+/OH− conductivity, (iii) H+/OH− fluxes are not governed by lipid order and (iv) chol can inhibit or promote H+/OH− permeability depending on the total lipid environment. Theories of proton permeation are discussed in the light of these results.

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cADP-ribose activates reconstituted ryanodine receptors from coronary arterial smooth muscle

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2001.280.1.h208 ◽

2001 ◽

Vol 280 (1) ◽

pp. H208-H215 ◽

Cited By ~ 41

Author(s):

Pin-Lan Li ◽

Wang-Xian Tang ◽

Hector H. Valdivia ◽

Ai-Ping Zou ◽

William B. Campbell

Keyword(s):

Smooth Muscle ◽

Lipid Bilayers ◽

Ryanodine Receptors ◽

Fold Increase ◽

Ruthenium Red ◽

Dependent Manner ◽

High Concentration ◽

Open Probability ◽

Arterial Smooth Muscle ◽

Concentration Dependent Manner

The present study was designed to test the hypothesis that cADP-ribose (cADPR) increases Ca2+release through activation of ryanodine receptors (RYR) on the sarcoplasmic reticulum (SR) in coronary arterial smooth muscle cells (CASMCs). We reconstituted RYR from the SR of CASMCs into planar lipid bilayers and examined the effect of cADPR on the activity of these Ca2+ release channels. In a symmetrical cesium methanesulfonate configuration, a 245 pS Cs+ current was recorded. This current was characterized by the formation of a subconductance and increase in the open probability (NPo) of the channels in the presence of ryanodine (0.01–1 μM) and imperatoxin A (100 nM). A high concentration of ryanodine (50 μM) and ruthenium red (40–80 μM) substantially inhibited the activity of RYR/Ca2+ release channels. Caffeine (0.5–5 mM) markedly increased the NPo of these Ca2+release channels of the SR, but d- myo-inositol 1,4,5-trisphospate and heparin were without effect. Cyclic ADPR significantly increased the NPo of these Ca2+release channels of SR in a concentration-dependent manner. Addition of cADPR (0.01 μM) into the cis bath solution produced a 2.9-fold increase in the NPo of these RYR/Ca2+release channels. An eightfold increase in the NPo of the RYR/Ca2+ release channels (0.0056 ± 0.001 vs. 0.048 ± 0.017) was observed at a concentration of cADPR of 1 μM. The effect of cADPR was completely abolished by ryanodine (50 μM). In the presence of cADPR, Ca2+-induced activation of these channels was markedly enhanced. These results provide evidence that cADPR activates RYR/Ca2+ release channels on the SR of CASMCs. It is concluded that cADPR stimulates Ca2+ release through the activation of RYRs on the SR of these smooth mucle cells.

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Influence of Brain Gangliosides on the Formation and Properties of Supported Lipid Bilayers for Binding Kinetics Studies

10.26434/chemrxiv.7505330 ◽

2018 ◽

Author(s):

Luke Jordan ◽

Nathan Wittenberg

Keyword(s):

Lipid Bilayers ◽

Binding Kinetics ◽

Supported Lipid Bilayers ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Supported Lipid Bilayer ◽

Head Groups ◽

Lipid Diffusion ◽

Kinetics Of ◽

Brain Gangliosides

This is a comprehensive study of the effects of the four major brain gangliosides (GM1, GD1b, GD1a, and GT1b) on the adsorption and rupture of phospholipid vesicles on SiO2 surfaces for the formation of supported lipid bilayer (SLB) membranes. Using quartz crystal microbalance with dissipation monitoring (QCM-D) we show that gangliosides GD1a and GT1b significantly slow the SLB formation process, whereas GM1 and GD1b have smaller effects. This is likely due to the net ganglioside charge as well as the positions of acidic sugar groups on ganglioside glycan head groups. Data is included that shows calcium can accelerate the formation of ganglioside-rich SLBs. Using fluorescence recovery after photobleaching (FRAP) we also show that the presence of gangliosides significantly reduces lipid diffusion coefficients in SLBs in a concentration-dependent manner. Finally, using QCM-D and GD1a-rich SLB membranes we measure the binding kinetics of an anti-GD1a antibody that has similarities to a monoclonal antibody that is a hallmark of a variant of Guillain-Barre syndrome.

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Quercetin Is a Flavonoid Breast Cancer Resistance Protein Inhibitor with an Impact on the Oral Pharmacokinetics of Sulfasalazine in Rats

Pharmaceutics ◽

10.3390/pharmaceutics12050397 ◽

2020 ◽

Vol 12 (5) ◽

pp. 397

Author(s):

Yoo-Kyung Song ◽

Jin-Ha Yoon ◽

Jong Kyu Woo ◽

Ju-Hee Kang ◽

Kyeong-Ryoon Lee ◽

...

Keyword(s):

Breast Cancer ◽

Breast Cancer Resistance Protein ◽

Fold Increase ◽

Inhibitory Effect ◽

Dependent Manner ◽

Cancer Resistance ◽

Concentration Dependent Manner ◽

Resistance Protein

The potential inhibitory effect of quercetin, a major plant flavonol, on breast cancer resistance protein (BCRP) activity was investigated in this study. The presence of quercetin significantly increased the cellular accumulation and associated cytotoxicity of the BCRP substrate mitoxantrone in human cervical cancer cells (HeLa cells) in a concentration-dependent manner. The transcellular efflux of prazosin, a stereotypical BCRP substrate, was also significantly reduced in the presence of quercetin in a bidirectional transport assay using human BCRP-overexpressing cells; further kinetic analysis revealed IC50 and Ki values of 4.22 and 3.91 μM, respectively. Moreover, pretreatment with 10 mg/kg quercetin in rats led to a 1.8-fold and 1.5-fold increase in the AUC8h (i.e., 44.5 ± 11.8 min∙μg/mL vs. 25.7 ± 9.98 min∙μg/mL, p < 0.05) and Cmax (i.e., 179 ± 23.0 ng/mL vs. 122 ± 23.2 ng/mL, p < 0.05) of orally administered sulfasalazine, respectively. Collectively, these results provide evidence that quercetin acts as an in vivo as well as in vitro inhibitor of BCRP. Considering the high dietary intake of quercetin as well as its consumption as a dietary supplement, issuing a caution regarding its food–drug interactions should be considered.

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Calcitonin Induces IL-6 Production via Both PKA and PKC Pathways in the Pituitary Folliculo-Stellate Cell Line

Endocrinology ◽

10.1210/endo.142.8.8328 ◽

2001 ◽

Vol 142 (8) ◽

pp. 3563-3569 ◽

Cited By ~ 11

Author(s):

Yoshimitsu Kiriyama ◽

Hiroyuki Tsuchiya ◽

Takeshi Murakami ◽

Kumi Satoh ◽

Yukiko Tokumitsu

Keyword(s):

Cell Line ◽

Stellate Cell ◽

Fold Increase ◽

Receptor Subtype ◽

Dependent Manner ◽

Calcitonin Receptor ◽

Rt Pcr ◽

Concentration Dependent Manner ◽

Mouse Pituitary ◽

Inhibitor Treatment

Abstract It has been demonstrated that calcitonin-binding sites are present in a variety of tissue types, including in the pituitary gland. Interleukin-6 (IL-6) is also produced in the pituitary and it regulates the secretion of various hormones. In this study, we examined the expression of the calcitonin receptor and the mechanism of IL-6 production induced by calcitonin in the pituitary folliculo-stellate cell line (TtT/GF). The mRNA of calcitonin receptor subtype C1a, but not that of C1b, was detected by RT-PCR in TtT/GF cells and in the normal mouse pituitary. Calcitonin increased cAMP accumulation and IL-6 production in a concentration-dependent manner in TtT/GF cells. As calcitonin activates the PKA and PKC pathways, we investigated the contributions of PKA and PKC to IL-6 production. IL-6 production was only slightly increased by either 8-bromo-cAMP (1 mm) or phorbol 12-myristate 13-acetate (100 nm) alone. However, IL-6 was synergistically induced in the presence of both 8-bromo-cAMP (1 mm) and phorbol 12myristate 13-acetate (100 nm). Furthermore, calcitonin-induced IL-6 production was completely suppressed by H-89 (PKA inhibitor) or GF109203X (PKC inhibitor), indicating that the activation of both PKA and PKC is necessary for calcitonin-induced IL-6 production. On the other hand, pertussis toxin (Gi/Go signaling inhibitor) treatment achieved an approximately 9-fold increase in calcitonin-induced IL-6 production. These results show that calcitonin-stimulated IL-6 production is mediated via both PKA- and PKC-signaling pathways, whereas calcitonin also suppresses IL-6 production by activating Gi/Go proteins in folliculo-stellate cells.

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Angiotensin II-Induced Mitogen-Activated Protein Kinase Phosphatase-1 Expression in Bovine Adrenal Glomerulosa Cells: Implications in Mineralocorticoid Biosynthesis

Endocrinology ◽

10.1210/en.2007-0241 ◽

2007 ◽

Vol 148 (11) ◽

pp. 5573-5581 ◽

Cited By ~ 10

Author(s):

Andrés J. Casal ◽

Stéphane Ryser ◽

Alessandro M. Capponi ◽

Carine F. Wang-Buholzer

Keyword(s):

Angiotensin Ii ◽

Mapk Pathway ◽

Fold Increase ◽

Zona Glomerulosa ◽

Mitogen Activated Protein Kinase ◽

Receptor Subtype ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Aldosterone Production ◽

Glomerulosa Cells

Angiotensin II (AngII) stimulates aldosterone biosynthesis in the zona glomerulosa of the adrenal cortex. AngII also triggers the MAPK pathways (ERK1/2 and p38). Because ERK1/2 phosphorylation is a transient process, phosphatases could play a crucial role in the acute steroidogenic response. Here we show that the dual specificity (threonine/tyrosine) MAPK phosphatase-1 (MKP-1) is present in bovine adrenal glomerulosa cells in primary culture and that AngII markedly increases its expression in a time- and concentration-dependent manner (IC50 = 1 nm), a maximum of 548 ± 10% of controls being reached with 10 nm AngII after 3 h (n = 3, P < 0.01). This effect is completely abolished by losartan, a blocker of the AT1 receptor subtype. Moreover, this AngII-induced MKP-1 expression is reduced to 250 ± 35% of controls (n = 3, P < 0.01) in the presence of U0126, an inhibitor of ERK1/2 phosphorylation, suggesting an involvement of the ERK1/2 MAPK pathway in MKP-1 induction. Indeed, shortly after AngII-induced phosphorylation of ERK1/2 (220% of controls at 30 min), MKP-1 protein expression starts to increase. This increase is associated with a reduction in ERK1/2 phosphorylation, which returns to control values after 3 h of AngII challenge. Enhanced MKP-1 expression is essentially due to a stabilization of MKP-1 mRNA. AngII treatment leads to a 53-fold increase in phosphorylated MKP-1 levels and a doubling of MKP-1 phosphatase activity. Overexpression of MKP-1 results in decreased phosphorylation of ERK1/2 and aldosterone production in response to AngII stimulation. These results strongly suggest that MKP-1 is the specific phosphatase induced by AngII and involved in the negative feedback mechanism ensuring adequate ERK1/2-mediated aldosterone production in response to the hormone.

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Insulin stimulates Mg2+ uptake in mouse distal convoluted tubule cells

AJP Renal Physiology ◽

10.1152/ajprenal.1999.277.6.f907 ◽

1999 ◽

Vol 277 (6) ◽

pp. F907-F913 ◽

Cited By ~ 8

Author(s):

Long-Jun Dai ◽

Gordon Ritchie ◽

Brian W. Bapty ◽

Dirk Kerstan ◽

Gary A. Quamme

Keyword(s):

Kinase Inhibitor ◽

Insulin Receptors ◽

Distal Tubule ◽

Fold Increase ◽

Distal Convoluted Tubule ◽

Maximal Response ◽

Thick Ascending Limb ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Camp Formation

Insulin has been shown to be a magnesium-conserving hormone acting, in part, through stimulation of magnesium absorption within the thick ascending limb. Although the distal convoluted tubule possesses the most insulin receptors, it is unclear what, if any, actions insulin has in the distal tubule. The effects of insulin were studied on immortalized mouse distal convoluted tubule (MDCT) cells by measuring cellular cAMP formation with radioimmunoassays and Mg2+ uptake with fluorescence techniques using mag-fura 2. To assess Mg2+ uptake, MDCT cells were first Mg2+ depleted to 0.22 ± 0.01 mM by culturing in Mg2+-free media for 16 h and then placed in 1.5 mM MgCl2, and the changes in intracellular Mg2+ concentration ([Mg2+]i) were measured with microfluorescence. [Mg2+]i returned to basal levels, 0.53 ± 0.02 mM, with a mean refill rate, d([Mg2+]i)/d t, of 164 ± 5 nM/s. Insulin stimulated Mg2+ entry in a concentration-dependent manner with maximal response of 214 ± 12 nM/s, which represented a 30 ± 5% increase in the mean uptake rate above control values. This was associated with a 2.5-fold increase in insulin-mediated cAMP generation (52 ± 3 pmol ⋅ mg protein−1 ⋅ 5 min−1). Genistein, a tyrosine kinase inhibitor, diminished insulin-stimulated Mg2+ uptake (169 ± 11 nM/s), but did not change insulin-mediated cAMP formation (47 ± 5 pmol ⋅ mg protein−1 ⋅ 5 min−1). PTH stimulates Mg2+ entry, in part, through increases in cAMP formation. Insulin and PTH increase Mg2+ uptake in an additive fashion. In conclusion, insulin mediates Mg2+ entry, in part, by a genistein-sensitive mechanism and by modifying hormone-responsive transport. These studies demonstrate that insulin stimulates Mg2+ uptake in MDCT cells and suggest that insulin acts in concert with other peptide and steroid hormones to control magnesium conservation in the distal convoluted tubule.

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Kinetic and physicochemical characteristics of an endogenous inhibitor to progesterone–receptor binding in rat placental cytosol

Biochemical Journal ◽

10.1042/bj1990371 ◽

1981 ◽

Vol 199 (2) ◽

pp. 371-381 ◽

Cited By ~ 6

Author(s):

T F Ogle

Keyword(s):

Progesterone Receptor ◽

Receptor Binding ◽

Binding Sites ◽

Fold Increase ◽

Dissociation Rate ◽

Energy Of Activation ◽

Gel Chromatography ◽

Dependent Manner ◽

Endogenous Inhibitor ◽

Concentration Dependent Manner

This study describes the kinetic behaviour and physicochemical aspects of an endogenous inhibitor of progesterone--receptor binding in trophoblast cytosol from day-12 embryos. The progesterone cytosol receptor was partially purified and isolated from the inhibitor as the 0--50%-satd. (NH4)2SO4 fraction. The inhibitory substance was shown to reside in the 50--70%-satd. (NH4)2SO4 fraction. Equilibration of the inhibitor preparation with the receptor fraction increased the Kapp.D of the ligand--receptor binding reaction in a concentration-dependent manner (26 +/- 3-fold increase in Kapp.D per mg of protein of the (NH4)2SO4 fraction, n = 16). However, the inhibitor did not alter the concentration of binding sites. Studies of other physicochemical aspects of the inhibitor showed it to be non-diffusible, excluded from Sephadex G-25, stable at 35 degrees C for 30 min, but irreversibly denatured at 70 degrees C for 30 min. The Stokes′ radius was estimated by gel chromatography to be 2.8 +/- 0.11 nm (n = 5). Inhibitory activity was destroyed by HgCl2, suggesting that disulphide bridges play an essential role in the biological activity of this molecule. The inhibitor is a macromolecule which does not bind progesterone and differs from albumin. The kinetic mechanism by which the inhibitor enhanced Kapp.D was investigated by measuring association and dissociation rate constants and the energy of activation (Ea) for each reaction. The association rate (k+1) for progesterone and receptor was (1.3 +/- 0.2) x 10(4) M-1 . s-1 but declined to (0.4 +/- 0.1) x 10(4) M-1 . s-1 (n = 5) when exposed to the inhibitor (P less than 0.01). The dissociation rate (k-1) was (3.2 +/- 0.6) x 10(-5) s-1 for progesterone--receptor complex and was unchanged by the inhibitor. The Ea for the association of complex was 33.6 +/- 4.2 kJ/mol and was increased to 63.0 +/- 8.4 kJ/mol by the inhibitor (P less than 0.05). The Ea of dissociation was unaltered. Thus, an inhibitor is present in trophoblast cytosol which specifically enhances Kapp.D without altering availability of binding sites. The mode of action of inhibitor is to increase the energy of activation for association of complex without influencing the dissociation reaction.

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A long-lasting, plasmin-activatable thrombin inhibitor aids clot lysis in vitro and does not promote bleeding in vivo

Thrombosis and Haemostasis ◽

10.1160/th08-08-0535 ◽

2009 ◽

Vol 101 (05) ◽

pp. 867-877 ◽

Cited By ~ 15

Author(s):

Louise Eltringham-Smith ◽

Sharon Gataiance ◽

Varsha Bhakta ◽

William Sheffield

Keyword(s):

Chimeric Protein ◽

Fold Increase ◽

Thrombin Inhibitor ◽

Clot Lysis ◽

Dependent Manner ◽

Recombinant Hirudin ◽

Concentration Dependent Manner ◽

Shed Blood

SummaryThe leech protein hirudin is a potent inhibitor of thrombin, but clinical use of recombinant hirudin is restricted by haemorrhagic risks, and complicated by hirudin’s rapid clearance from the circulation. We previously employed albumin fusion to slow hirudin variant 3 (HV3) clearance. In this study, we hypothesized that reconfiguration of the chimera, appending human serum albumin (HSA) to the N-terminus of HV3, with an intervening plasmin cleavage site, would create a slowly cleared, plasmin-activatable HV3. Potential plasmin cleavage sites were screened by expression in Escherichia coli, interposed between glutathione sulfotransferase and HV3 domains. The most reactive sequence (GSGIYR-ITY) was recreated in C-terminally His-tagged albumin fusion protein HSACHV3, expressed in Pichia pastoris yeast and purified by nickel-chelate affinity chromatography. HSACHV3 showed no thrombin inhibitory activity in the absence of plasmin, but liberated active HV3 in a time- and concentration-dependent manner in its presence. In a discontinuous clot assay involving clot-bound thrombin, HSACHV3 assisted clot lysis by limiting clot extension in a tPA- and concentration-dependent manner. Similar results were obtained in plasma at higher concentrations of HSACHV3. The chimeric protein exhibited much slower clearance in mice than unfused HV3, and indistinguishable pharmacokinetics from unfused recombinant HSA. In a mouse tail transection bleeding model, doses of HSACHV3 identical to those of HV3 that elicited a four-fold increase in the volume of shed blood were without effect. Our results suggest that HSACHV3 is a fully latent, plasmin activatable, long-lasting hirudin, of potential benefit in thrombotic disorders resistant to natural or pharmacological clot lysis.

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Influence of Brain Gangliosides on the Formation and Properties of Supported Lipid Bilayers for Binding Kinetics Studies

10.26434/chemrxiv.7505330.v1 ◽

2018 ◽

Author(s):

Luke Jordan ◽

Nathan Wittenberg

Keyword(s):

Lipid Bilayers ◽

Binding Kinetics ◽

Supported Lipid Bilayers ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Supported Lipid Bilayer ◽

Head Groups ◽

Lipid Diffusion ◽

Kinetics Of ◽

Brain Gangliosides

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Zinc Alters Fibrin Ultrastructure

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651065 ◽

1987 ◽

Vol 57 (01) ◽

pp. 073-076 ◽

Cited By ~ 17

Author(s):

Gerard Marx ◽

Pierre Hopmeier ◽

Dorit Gurfel

Keyword(s):

Cross Section ◽

Electron Micrographs ◽

Fibre Diameter ◽

Fold Increase ◽

Fibrin Clot ◽

Dependent Manner ◽

Clotting Time ◽

Concentration Dependent Manner ◽

Sem Micrographs ◽

Fibrin Monomer

SummaryTurbidimetric studies indicate that Zn(II) accelerates fibrin gelation [decreases clotting time (CT)] and increases maximal fibrin clot turbidity. For any given level of fibrinogen (0.2-2.6 mg/ ml), the relative fibrin turbidity of thrombin-induced clots increases with Zn(II) in a concentration dependent manner. Zinc-associated turbidity increases are also observed in the presence of 2 mM Ca(II). With citrate, similar turbidity increases are observed, though at higher cation levels. Thus, turbidimetry indicates that the gel formed with Zn(II) is coarser, or has thicker fibre strands. SEM micrographs confirm that fibre thickness ranges from 260 Å to 2600 Å, when Zn(II) levels range from 0-50 uM. With citrate, TEM micrographs reveal amore than 20 x fold increase in fibre diameter (100 Å->2000 Å) with higher Zn(II) (<1 mM) levels. Based on a fibrin monomer cross-section of ~60 Å, the electron micrographs indicate that depending on the Zn(II) levels, fibrin strands are composed of between 2 to 40 monomeric fibrin molecules. Thus, at physiologically relevant levels, Zn(II) can drastically modulate fibrin ultrastructure.

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