Vasopressin (AVP) and oxytocin (OT) receptors (nomenclature as recommended by NC-IUPHAR [98]) are activated by the endogenous cyclic nonapeptides vasopressin and oxytocin. These peptides are derived from precursors which also produce neurophysins (neurophysin I for oxytocin; neurophysin II for vasopressin). Vasopressin and oxytocin differ at only 2 amino acids (positions 3 and 8). There are metabolites of these neuropeptides that may be biologically active [71].

The enzyme acyl-CoA:cholesterol acyltransferase (ACAT) is normally localized in the endoplasmic reticulum (ER) where it can esterify cholesterol for storage in lipid droplets and/or the formation of lipoproteins. Here, we report that ACAT can translocate from the ER into vesicular structures in response to different ACAT inhibitors. The translocation was fast (within minutes), reversible and occurred in different cell types. Interestingly, oleic acid was able to fasten the re-translocation from vesicles back into the reticular ER network. The process of ACAT translocation could also be induced by cyclodextrins, cholesterol, lanosterol (but not 4-cholestene-3 one), 25-hydroxycholesterol, and by certain stress stimuli such as hyperosmolarity (sucrose treatment), temperature change, or high-density cultivation. In vitro esterification showed that ACAT remains fully active after it has been translocated to vesicles in response to hyperosmotic sucrose treatment of the cells. The translocation process was not accompanied by changes in the electrophoretic mobility of ACAT, even after chemical crosslinking. Interestingly, the protein synthesis inhibitor cycloheximide showed a stimulating effect on ACAT activity and prevented the translocation of ACAT from the ER into vesicles.

Vasopressin (AVP) and oxytocin (OT) receptors (nomenclature as recommended by NC-IUPHAR [94]) are activated by the endogenous cyclic nonapeptides vasopressin and oxytocin. These peptides are derived from precursors which also produce neurophysins (neurophysin I for oxytocin; neurophysin II for vasopressin). Vasopressin and oxytocin differ at only 2 amino acids (positions 3 and 8). There are metabolites of these neuropeptides that may be biologically active [69].

We investigated stability and change of plasma and urinary oxytocin as well as OXTR DNA methylation patterns through psychotherapy. Furthermore, we explored the potential impact of inpatient psychotherapy on oxytocin-related biomarkers and vice versa by differentiating patients who remitted from depression versus non-remitters. Blood and urine samples were taken from 85 premenopausal women (aged 19–52), 43 clinically depressed patients from a psychosomatic inpatient unit, and 42 healthy control subjects matched for age and education at two points of time. Serum and urine oxytocin were measured using standard ELISA, and DNA methylation of the OXTR gene was assessed using bisulfite sequencing at the time of admission (baseline) and at discharge and from controls at matched time points. Oxytocin plasma levels were not associated with depression and were influenced by neither time in healthy controls nor psychotherapy in patients. Non-remitting depressed patients had significantly lower oxytocin urine levels before and after psychotherapy treatment. We found significantly lower exon 1 OTXR methylation in depressed patients over time and these differences were driven by patients remitting due to psychotherapy. A reverse pattern — higher levels of methylation in remitters — was found for exon 2 OXTR DNA methylation. Plasma oxytocin, urinary oxytocin, and OXTR DNA methylation patterns were intrapersonally relatively stable. OXTR-related factors were seemingly unaffected by inpatient psychotherapeutic treatment, but we found significant differences between remitting and non-remitting patients in urinary oxytocin and OXTR DNA methylation. If replicated, this suggests that OXTR-related markers may predict inpatient treatment outcomes of clinically depressed patients.

The oxytocin receptor, a class A G protein coupled receptor (GPCR), is essentially involved in the physiology of reproduction. Two parameters are crucially important to support high-affinity agonist binding of the receptor: Mg2+ and cholesterol, both acting as positive modulators. Using displacement assays with a high-affinity fluorescent antagonist (OTAN-A647), we now show that sodium functions as a negative allosteric modulator of the oxytocin receptor. In membranes from HEK293 cells stably expressing the oxytocin receptor, oxytocin binding occurred with about 15-fold lower affinity when sodium chloride was increased from 0 to 300 mM, whereas antagonist binding remained largely unchanged. The effect was concentration-dependent, sodium-specific, and it was also observed for oxytocin receptors endogenously expressed in Hs578T breast cancer cells. A conserved Asp (Asp 85) is known to stabilize the sodium binding site in other GCPRs. Mutations of this residue into Ala or Asn are known to yield non-functional oxytocin receptors. When Asp 85 was exchanged for Glu, most of the oxytocin receptors were localized in intracellular structures, but a faint plasma membrane labeling with OTAN-A647 and the appearance of oxytocin-induced calcium responses indicated that these receptors were functional. However, a sodium effect was not detectable for the mutant D85E oxytocin receptors. Thus, the oxytocin receptor is allosterically controlled by sodium similar to other GPCRs, but it behaves differently concerning the involvement of the conserved Asp 85. In case of the oxytocin receptor, Asp 85 is obviously essential for proper localization in the plasma membrane.

We introduce the novel fluorescent cholesterol probe RChol in which a sulforhodamine group is linked to the sixth carbon atom of the steroid backbone of cholesterol. The same position has recently been selected to generate the fluorescent reporter 6-dansyl-cholestanol (DChol) and the photoreactive 6-azi-cholestanol. In comparison with DChol, RChol is brighter, much more photostable, and requires less energy for excitation, i.e. favorable conditions for microscopical imaging. RChol easily incorporates into methyl-β-cyclodextrin forming a water-soluble inclusion complex that acts as an efficient sterol donor for cells and membranes. Like cholesterol, RChol possesses a free 3′OH group, a prerequisite to undergo intracellular esterification. RChol was also able to support the growth of cholesterol auxotrophic cells and can therefore substitute for cholesterol as a major component of the plasma membrane. According to subcellular fractionation, slight amounts of RChol (~12%) were determined in low-density Triton-insoluble fractions whereas the majority of RChol was localized in non-rafts fractions. In phase-separated giant unilamellar vesicles, RChol preferentially partitions in liquid-disordered membrane domains. Intracellular RChol was transferred to extracellular sterol acceptors such as high density lipoproteins in a dose-dependent manner. Unlike DChol, RChol was not delivered to the cholesterol storage pathway. Instead, it translocated to endosomes/lysosomes with some transient contacts to peroxisomes. Thus, RChol is considered as a useful probe to study the endosomal/lysosomal pathway of cholesterol.

G protein coupled receptors (GPCRs) form the largest receptor superfamily in eukaryotic cells. Owing to their seven transmembrane helices, large parts of these proteins are embedded in the cholesterol-rich plasma membrane bilayer. Thus, GPCRs are always in proximity to cholesterol. Some of them are functionally dependent on the specific presence of cholesterol. Over the last years, enormous progress on receptor structures has been achieved. While lipophilic ligands other than cholesterol have been shown to bind either inside the helix bundle or at the receptor-lipid interface, the binding site of cholesterol was either a single transmembrane helix or a groove between two or more transmembrane helices. A clear preference for one of the two membrane leaflets has not been observed. Not surprisingly, many hydrophobic residues (primarily leucine and isoleucine) were found to be involved in cholesterol binding. In most cases, the rough β-face of cholesterol contacted the transmembrane helix bundle rather than the surrounding lipid matrix. The polar hydroxy group of cholesterol was localized near the water-membrane interface with potential hydrogen bonding to residues in receptor loop regions. Although a canonical motif, designated as CCM site, was detected as a specific cholesterol binding site in case of the β2AR, this site was not found to be occupied by cholesterol in other GPCRs possessing the same motif. Cholesterol-receptor interactions can increase the compactness of the receptor structure and are able to enhance the conformational stability towards active or inactive receptor states. Overall, all current data suggest a high plasticity of cholesterol interaction sites in GPCRs.

During first merogony Eimeria bovis forms large macromeronts in endothelial host cells containing >120 000 merozoites I. During multiplication, large amounts of cholesterol are indispensable for the enormous offspring membrane production. Cholesterol auxotrophy was proven for other apicomplexan parasites. Consequently they scavenge cholesterol from their host cell apparently in a parasite-specific manner. We here analyzed the influence of E. bovis infection on endothelial host cell cholesterol metabolism and found considerable differences to other coccidian parasites. Overall, free cholesterol significantly accumulated in E. bovis infected host cells. Furthermore, a striking increase of lipid droplet formation was observed within immature macromeronts. Artificial host cell lipid droplet enrichment significantly improved E. bovis merozoite I production confirming the key role of lipid droplet contents for optimal parasite proliferation. The transcription of several genes being involved in both, cholesterol de novo biosynthesis and low density lipoprotein-(LDL) mediated uptake, was significantly up-regulated at a time in infected cells suggesting a simultaneous exploitation of these two cholesterol acquisition pathways. E. bovis scavenges LDL-derived cholesterol apparently through significantly increased levels of surface LDL receptor abundance and LDL binding to infected cells. Consequently, LDL supplementation significantly improved parasite replication. The up-regulation of the oxidized LDL receptor 1 furthermore identified this scavenger receptor as a key molecule in parasite-triggered LDL uptake. Moreover, cellular cholesterol processing was altered in infected cells as indicated by up-regulation of cholesterol-25-hydroxylase and sterol O-acyltransferase. Overall, these results show that E. bovis considerably exploits the host cell cholesterol metabolism to guarantee its massive intracellular growth and replication.

Melittin, the major component of the bee venom, is an amphipathic, cationic peptide with a wide spectrum of biological properties that is being considered as an anti-inflammatory and anti-cancer agent. It modulates multiple cellular functions but the underlying mechanisms are not clearly understood. Here, we report that melittin activates disintegrin-like metalloproteases (ADAMs) and that downstream events likely contribute to the biological effects evoked by the peptide. Melittin stimulated the proteolysis of ADAM10 and ADAM17 substrates in human neutrophil granulocytes, endothelial cells and murine fibroblasts. In human HaCaT keratinocytes, melittin induced shedding of the adhesion molecule E-cadherin and release of TGF-α, which was accompanied by transactivation of the EGF receptor and ERK1/2 phosphorylation. This was followed by functional consequences such as increased keratinocyte proliferation and enhanced cell migration. Evidence is provided that ATP release and activation of purinergic P2 receptors are involved in melittin-induced ADAM activation. E-cadherin shedding and EGFR phosphorylation were dose-dependently reduced in the presence of ATPases or P2 receptor antagonists. The involvement of P2 receptors was underscored in experiments with HEK cells, which lack the P2X7 receptor and showed strikingly increased response to melittin stimulation after transfection with this receptor. Our study provides new insight into the mechanism of melittin function which should be of interest particularly in the context of its potential use as an anti-inflammatory or anti-cancer agent.

This chapter contains sections titled: Introduction Presence of Cholesterol-Binding Domains? Presence of Cholesterol-Rich Microdomains? Alterations in Protein Function Induced by Changes in the Cholesterol Content in Membranes/Cells? Alterations in Receptor Function, Following Substitution of Cholesterol by Sterol Analogs? Correlation of Receptor Function with Membrane Parameters, E.G., Membrane Fluidity? Cholesterol-Binding Molecules as Functional “Cholesterol Competitors”? Cholesterol Requirement for Heterologous (OVER) Expression, Solubilization, and Purification of Receptors? Direct Interaction of the Receptor with Labeled Cholesterol Probes? Concluding Remarks References

The disintegrin-metalloproteinases ADAM10 and ADAM17 mediate the release of several cell signaling molecules and cell adhesion molecules such as vascular endothelial cadherin or L-selectin affecting endothelial permeability and leukocyte transmigration. Dysregulation of ADAM activity may contribute to the pathogenesis of vascular diseases, but the mechanisms underlying the control of ADAM functions are still incompletely understood. Atherosclerosis is characterized by lipid plaque formation and local accumulation of unsaturated free fatty acids (FFA). Here, we show that unsaturated FFA increase ADAM-mediated substrate cleavage. We demonstrate that these alterations are not due to genuine changes in enzyme activity, but correlate with changes in membrane fluidity as revealed by measurement of 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy and fluorescence recovery after photobleaching analyses. ELISA and immunoblot experiments conducted with granulocytes, endothelial cells, and keratinocytes revealed rapid increase of ectodomain shedding of ADAM10 and ADAM17 substrates upon membrane fluidization. Large amounts of unsaturated FFA may be liberated from cholesteryl esters in LDL that is entrapped in atherosclerotic lesions. Incubation of cells with thus modified LDL resulted in rapid cleavage of ADAM substrates with corresponding functional consequences on cell proliferation, cell migration, and endothelial permeability, events of high significance in atherogenesis. We propose that FFA represent critical regulators of ADAM function that may assume relevance in many biological settings through their influence on mobility of enzyme and substrate in lipid bilayers.

The oxytocin receptor specifically requires cholesterol to maintain and stabilize its high-affinity agonist binding. Here, we applied a receptor chimeric approach to coarsely localize the cholesterol binding domain of the oxytocin receptor. During these studies, we identified the specific dependence on cholesterol as a common property of the oxytocin-vasopressin receptor family. We asked whether the oxytocin receptor maintains or loses its cholesterol dependence when parts of the receptor are exchanged by the corresponding fragments of the cholecystokinin receptor that does not show a specific cholesterol dependence. One of the chimeric receptors revealed full oxytocin binding activity, was capable of signal transduction, and its cholesterol dependence was nearly indistinguishable from that of the oxytocin receptor itself. The data suggest that at least the C-terminal segments of the oxytocin receptor including transmembrane domains 6 and 7 do not participate in cholesterol binding. Thus, the binding sites for the modulator cholesterol and for the agonist oxytocin are therefore most likely colocalized in the N-terminal segment of the oxytocin receptor.

Recent studies suggest that cholesterol binding is widespread among GPCRs (G-protein-coupled receptors). In the present study, we analysed putative cholesterol-induced changes in the OTR [OT (oxytocin) receptor], a prototype of cholesterol-interacting GPCRs. For this purpose, we have created recombinant OTRs that are able to bind two small-sized fluorescence-labelled ligands simultaneously. An OTR antagonist was chosen as one of the ligands. To create a second ligand-binding site, a small-sized α-BTB (bungarotoxin binding) site was inserted at the N-terminus or within the third extracellular loop of the OTR. All receptor constructs were functionally active and bound both ligands with high affinity in the nanomolar range. Measurements of the quenching behaviour, fluorescence anisotropy and energy transfer of both receptor-bound ligands were performed to monitor receptor states at various cholesterol concentrations. The quenching studies suggested no major changes in the molecular environment of the fluorophores in response to cholesterol. The fluorescence anisotropy data indicated that cholesterol affects the dynamics or orientation of the antagonist. The energy transfer efficiency between both ligands clearly increased with increasing cholesterol. Overall, cholesterol induced both a changed orientation and a decreased distance of the receptor-bound ligands, suggesting a more compact receptor state in association with cholesterol.

Cholesterol is a multifunctional lipid in eukaryotic cells. It regulates the physical state of the phospholipid bilayer, is crucially involved in the formation of membrane microdomains, affects the activity of many membrane proteins, and is the precursor for steroid hormones and bile acids. Thus, cholesterol plays a profound role in the physiology and pathophysiology of eukaryotic cells. The cholesterol molecule has achieved evolutionary perfection to fulfill its different functions in membrane organization. Here, we review basic approaches to explore the interaction of cholesterol with proteins, with a particular focus on the high diversity of fluorescent and photoreactive cholesterol probes available today.

Reactive oxygen species (ROS) have important roles as signaling molecules in the regulation of a variety of biological processes. On the other hand, chronic oxidative stress exerted by ROS is widely considered a causative factor in aging. Therefore, cells need to be able to adapt to a chronic oxidative challenge and do so to a certain cell-type-specific extent. Recently, we have shown in oxidative-stress-resistant cell lines, HT22H2O2 and HT22Glu, derived from the neuronal cell line HT22 by chronic exposure to sublethal concentrations of H2O2 and glutamate, that, in addition to the known antioxidant defense mechanisms, e.g., activation of antioxidant enzymes or up-regulation of heat-shock proteins, oxidative stress resistance depends on the composition of cellular membranes. Here, we extend our previous investigations and report increased membrane fluidity in HT22H2O2 and HT22Glu cells compared to the parental HT22WT cells. The increased membrane fluidity correlates with a redistribution of cholesterol, sphingomyelin, and membrane-associated proteins involved in APP processing between detergent-resistant and detergent-soluble membrane subdomains. The altered membrane properties were associated with drastic changes in the metabolism of the Alzheimer disease-associated amyloid precursor protein (APP), particularly toward enhanced production of soluble APPα, which is a known neuroprotective factor. Thus our -data provide a link between chronic oxidative stress, alterations in membrane fluidity and composition of membrane subdomains, stress adaptation, and APP processing.

Progesterone non-genomically attenuates the calcium signaling of the human oxytocin receptor and several other Gαq protein-coupled receptors. High progesterone concentrations are found in the endometrium during pregnancy opposing the responsiveness of the underlying myometrium to labor-inducing hormones. Here, we demonstrate that within minutes, progesterone inhibits oxytocin- and bradykinin-induced contractions of rat uteri, calcium responses induced by platelet-activating factor in the human endometrial cell line MFE-280, and oxytocin-induced calcium signals in PHM1-31 immortalized pregnant human myometrial cells. Using human embryonic kidney (HEK293) cells as model system, we analyzed the molecular mechanisms underlying these effects. Our data indicate that progesterone rapidly depletes intracellular calcium stores. The resulting desensitization of the cells might contribute to the quiescence of the uterus during pregnancy.

Cholesterol is a major constituent of the plasma membrane in eukaryotic cells. It regulates the physical state of the phospholipid bilayer and is crucially involved in the formation of membrane microdomains. Cholesterol also affects the activity of several membrane proteins, and is the precursor for steroid hormones and bile acids. Here, methods are described that are used to explore the binding and/or interaction of proteins to cholesterol. For this purpose, a variety of cholesterol probes bearing radio-, spin-, photoaffinity- or fluorescent labels are currently available. Examples of proven cholesterol binding molecules are polyene compounds, cholesterol-dependent cytolysins, enzymes accepting cholesterol as substrate, and proteins with cholesterol binding motifs. Main topics of this report are the localization of candidate membrane proteins in cholesterol-rich microdomains, the issue of specificity of cholesterol- protein interactions, and applications of the various cholesterol probes for these studies.

Fluorescent analogues of cholesterol offer a powerful approach for monitoring cholesterol behavior in membranes because of their high sensitivity, suitable time resolution, and multiplicity of measurable parameters. In this work, we have monitored the orientation and dynamics of a novel fluorescent cholesterol probe, 6-dansylcholestanol (DChol), in membranes of different phase type utilizing sensitive fluorescence techniques including the red-edge excitation shift (REES) approach. Our results show that fluorescence emission maximum, anisotropy, and lifetime of DChol are dependent on the phase of the membrane. Interestingly, DChol exhibits significant red-edge excitation shift (REES) that appear to depend on the phase of the membrane. Analysis of membrane penetration depth by the parallax method shows that the dansyl group of DChol is localized at the interfacial region of the membrane ( approximately 15.6 A from the center of the bilayer). This is in excellent agreement with the previously reported location of cholesterol in fluid-phase membranes. We propose that DChol could be a potentially useful cholesterol analogue in future studies of model and biological membranes.

Chronic oxidative stress has been causally linked to several neurodegenerative disorders. As sensitivity for oxidative stress greatly differs between brain regions and neuronal cell types, specific cellular mechanisms of adaptation to chronic oxidative stress should exist. Our objective was to identify molecular mechanisms of adaptation of neuronal cells after applying chronic sublethal oxidative stress. We demonstrate that cells resistant to oxidative stress exhibit altered cholesterol and sphingomyelin metabolisms. Stress-resistant cells showed reduced levels of molecules involved in cholesterol trafficking and intracellular accumulation of cholesterol, cholesterol precursors, and metabolites. Moreover, stress-resistant cells exhibited reduced SMase activity. The altered lipid metabolism was associated with enhanced autophagy. Treatment of stress-resistant cells with neutral SMase reversed the stress-resistant phenotype, whereas it could be mimicked by treatment of neuronal cells with a specific inhibitor of neutral SMase. Analysis of hippocampal and cerebellar tissue of mouse brains revealed that the obtained cell culture data reflect the in vivo situation. Stress-resistant cells in vitro showed similar features as the less vulnerable cerebellum in mice, whereas stress-sensitive cells resembled the highly sensitive hippocampal area. These findings suggest an important role of the cell type-specific lipid profile for differential vulnerabilities of different brain areas toward chronic oxidative stress.