The neurohypophysial peptide oxytocin (OT) and OT-like hormones facilitate reproduction in all vertebrates at several levels. The major site of OT gene expression is the magnocellular neurons of the hypothalamic paraventricular and supraoptic nuclei. In response to a variety of stimuli such as suckling, parturition, or certain kinds of stress, the processed OT peptide is released from the posterior pituitary into the systemic circulation. Such stimuli also lead to an intranuclear release of OT. Moreover, oxytocinergic neurons display widespread projections throughout the central nervous system. However, OT is also synthesized in peripheral tissues, e.g., uterus, placenta, amnion, corpus luteum, testis, and heart. The OT receptor is a typical class I G protein-coupled receptor that is primarily coupled via G(q) proteins to phospholipase C-beta. The high-affinity receptor state requires both Mg(2+) and cholesterol, which probably function as allosteric modulators. The agonist-binding region of the receptor has been characterized by mutagenesis and molecular modeling and is different from the antagonist binding site. The function and physiological regulation of the OT system is strongly steroid dependent. However, this is, unexpectedly, only partially reflected by the promoter sequences in the OT receptor gene. The classical actions of OT are stimulation of uterine smooth muscle contraction during labor and milk ejection during lactation. While the essential role of OT for the milk let-down reflex has been confirmed in OT-deficient mice, OT's role in parturition is obviously more complex. Before the onset of labor, uterine sensitivity to OT markedly increases concomitant with a strong upregulation of OT receptors in the myometrium and, to a lesser extent, in the decidua where OT stimulates the release of PGF(2 alpha). Experiments with transgenic mice suggest that OT acts as a luteotrophic hormone opposing the luteolytic action of PGF(2 alpha). Thus, to initiate labor, it might be essential to generate sufficient PGF(2 alpha) to overcome the luteotrophic action of OT in late gestation. OT also plays an important role in many other reproduction-related functions, such as control of the estrous cycle length, follicle luteinization in the ovary, and ovarian steroidogenesis. In the male, OT is a potent stimulator of spontaneous erections in rats and is involved in ejaculation. OT receptors have also been identified in other tissues, including the kidney, heart, thymus, pancreas, and adipocytes. For example, in the rat, OT is a cardiovascular hormone acting in concert with atrial natriuretic peptide to induce natriuresis and kaliuresis. The central actions of OT range from the modulation of the neuroendocrine reflexes to the establishment of complex social and bonding behaviors related to the reproduction and care of the offspring. OT exerts potent antistress effects that may facilitate pair bonds. Overall, the regulation by gonadal and adrenal steroids is one of the most remarkable features of the OT system and is, unfor...

Biochemical, epidemiological, and genetic findings demonstrate a link between cholesterol levels, processing of the amyloid precursor protein (APP), and Alzheimer's disease. In the present report, we identify the ␣-secretase ADAM 10 (a disintegrin and metalloprotease) as a major target of the cholesterol effects on APP metabolism. Treatment of various peripheral and neural cell lines with either the cholesterol-extracting agent methyl-␤-cyclodextrin or the hydroxymethyl glutaryl-CoA reductase inhibitor lovastatin resulted in a drastic increase of secreted ␣-secretase cleaved soluble APP. This strong stimulatory effect was in the range obtained with phorbol esters and was further increased in cells overexpressing ADAM 10. In cells overexpressing APP, the increase of ␣-secretase activity resulted in a decreased secretion of A␤ peptides. Several mechanisms were elucidated as being the basis of enhanced ␣-secretase activity: increased membrane fluidity and impaired internalization of APP were responsible for the effect observed with methyl-␤-cyclodextrin; treatment with lovastatin resulted in higher expression of the ␣-secretase ADAM 10. Our results demonstrate that cholesterol reduction promotes the nonamyloidogenic ␣-secretase pathway and the formation of neuroprotective ␣-secretase cleaved soluble APP by several mechanisms and suggest approaches to prevention of or therapy for Alzheimer's disease.A myloid-␤ peptides (A␤), the principal proteinaceous components of amyloid plaques in brains of Alzheimer's disease (AD) patients, are derived from proteolytic cleavage of the amyloid precursor protein (APP), a type I integral membrane protein that is ubiquitously expressed. Both during and after its transport through the secretory pathway to the surface of cultured cells, a fraction of APP molecules undergoes specific endoproteolytic cleavage, most frequently by a scission between amino acids 16 and 17 of the A␤ region (1). This principal secretory cleavage is effected by (a) protease(s) designated as ␣-secretase(s). Soluble N-terminal APP fragments of 105-125 kDa are released into vesicle lumens and from the cell surface; similar species are readily detected in human plasma and cerebrospinal fluid (2). Recently, evidence has been provided that members of the ADAM family (a disintegrin and metalloprotease) act as ␣-secretases (3-5). For ADAM 10, basal and protein kinase C-stimulated ␣-secretase activity and many properties expected for the proteolytic processing of APP have been found (4).The stimulation of ␣-secretase activity and an increase of ␣-secretase cleaved soluble APP (APPs␣) might be beneficial for the treatment of AD for several reasons. In principle, proteolytic cleavage of APP within the A␤ sequence precludes the formation of the amyloid peptides derived from alternative proteolysis of APP with the ␤-secretase cleaving at the N terminus and the ␥-secretase(s) at the C terminus of A␤ peptides (for a recent review of APP processing, see ref. 6). On the other hand, APPs␣ has trophic effects on cerebral neuro...

To investigate the effect of cholesterol on the oxytocin receptor function in myometrial membranes, we developed a new method to alter the membrane cholesterol content. Using a methyl-substituted beta-cyclodextrin, we were able to selectively deplete the myometrial plasma membrane of cholesterol. Vice versa, incubating cholesterol-depleted membranes with a preformed soluble cholesterol-methyl-beta-cyclodextrin complex restored the cholesterol content of the plasma membrane. Binding experiments showed that, with the removal of cholesterol from the membrane, the dissociation constant for [3H]oxytocin is enhanced 87-fold (from Kd = 1.5 nM to Kd = 131 nM), therefore shifting the oxytocin receptor from high to low affinity. Increasing the cholesterol content of the cholesterol-depleted membrane again restored the high-affinity binding (Kd = 1.2 nM). The presence of 0.1 mM GTP gamma S did not significantly change the number of high-affinity binding sites for [3H]oxytocin in native plasma membranes, in membranes depleted of cholesterol, and in plasma membranes with restored cholesterol content. The number of high-affinity binding sites for the oxytocin antagonist [3H]PrOTA was dependent in the same way on the cholesterol content as for [3H]oxytocin. Substitution of the membrane cholesterol with other steroids showed a strong dependence of the oxytocin receptor function on the structure of the cholesterol molecule. The detergent-solubilized oxytocin receptor was not saturable with [3H]oxytocin even at concentrations up to 10(-6) M of radioligand. Addition of the cholesterol-methyl-beta-cyclodextrin complex to the detergent-solubilized oxytocin receptor induced a saturation of the solubilized binding sites (Bmax = 0.98 pmol/mg) for oxytocin (Kd = 16 nM).(ABSTRACT TRUNCATED AT 250 WORDS)

The modulatory effect of cholesterol on the function of two structurally related peptide receptors, the oxytocin receptor and the brain cholecystokinin receptor in plasma membranes as well as in intact cells, was analyzed. Different approaches for cholesterol modification were applied: (i) depletion and reloading of cholesterol mediated by methyl-beta-cyclodextrin and cholesterol-methyl-beta-cyclodextrin, respectively, in a reversible manner; (ii) mild treatment of the plasma membranes with cholesterol oxidase under control of the membrane fluidity as measured by fluorescence anisotropy of diphenylhexatriene; and (iii) filipin pretreatment of membranes. The results allowed us to distinguish two mechanisms of cholesterol affecting the ligand-binding function of receptors: changes of the membrane fluidity as demonstrated for the cholecystokinin receptor, or a putatively specific cholesterol-receptor interaction as shown for the oxytocin receptor. This was confirmed in a structure-activity analysis with a variety of sterol analogues substituting for cholesterol in the membranes. While the agonist binding of the cholecystokinin receptor was supported by each of the tested steroids and was well correlated with the corresponding fluorescence anisotropy values, a stringent and unique requirement of the oxytocin receptor's affinity state for structural features of the sterol molecule was found. The molecular requirements differ both from those postulated for sterol-phospholipid interactions and from those known to be necessary for the functional activity of other proteins. The different behavior of both peptide receptors concerning the cholesterol dependence of their ligand binding was also present in vivo at the level of signal transduction. The results suggest that cholesterol can modulate receptor function by two distinct mechanisms, by changes of the membrane fluidity, and/or by a highly specific molecular interaction.

Cholesterol influences many of the biophysical properties of membranes and is nonrandomly distributed between cellular organelles, subdomains of membranes, and leaflets of the membrane bilayer. In combination with the high dynamics of cholesterol distribution, this offers many possibilities for regulation of membrane-embedded receptors. Depending on the receptor, cholesterol can have a strong influence on the affinity state, on the binding capacity, and on signal transduction. Most important, cholesterol may stabilize receptors in defined conformations related to their biological functions. This may occur by direct molecular interaction between cholesterol and receptors. In this review, we discuss the functional dependence of the nicotinic acetylcholine receptor as well as different G protein-coupled receptors on the presence of cholesterol.

No abstract

We have expressed a c-myc epitope-tagged human oxytocin receptor in the baculovirus/Sf9 cell system. The receptor was identified by SDS-PAGE and subsequent immunoblot as a approximately 50 kDa protein which decreased to about 44 kDa upon treatment with tunicamycin. Binding studies showed that the human oxytocin receptor was expressed in a low-affinity state (Kd = 215 nM; Bmax = 1.66 pmol/mg). After addition of cholesterol in the form of a soluble cholesterol-methyl-beta-cyclodextrin complex to the membranes, we obtained part of the human oxytocin receptor in its high-affinity state for oxytocin (Kd = 0.96 nM and Bmax = 318 fmol/mg of protein). In subsequent studies, we added the cholesterol-methyl-beta-cyclodextrin complex to the Sf9 cell culture medium at various times post infection. Binding analysis showed that this results in a more than 3-fold further increase in functional receptor binding sites of high-affinity state (Bmax = 1.08 pmol/mg). The cholesterol effect was dose-dependent, with an EC50 of about 50 microM cholesterol. Due to these findings, we determined the cholesterol and phospholipid content in purified Sf9 plasma membranes. The untreated naturally cholesterol auxotroph insect cells grown in medium with 2% fetal calf serum had a molar cholesterol/phospholipid ratio of about 0.04, which is approximately 20-fold lower than normally found in plasma membranes of higher eukaryotic cells. The high-affinity binding of the oxytocin receptor increased in parallel with the cholesterol levels present in the corresponding plasma membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Antidepressants are commonly supposed to enhance serotonergic and/or noradrenergic neurotransmission by inhibition of neurotransmitter reuptake through binding to the respective neurotransmitter transporters or through inhibition of the monoamine oxidase. Using the concentration-clamp technique and measurements of intracellular Ca 2 þ , we demonstrate that different classes of antidepressants act as functional antagonists at the human 5-HT 3A receptor stably expressed in HEK 293 cells and at endogenous 5-HT 3 receptors of rat hippocampal neurons and N1E-115 neuroblastoma cells. The tricyclic antidepressants desipramine, imipramine, and trimipramine, the serotonin reuptake inhibitor fluoxetine, the norepinephrine reuptake inhibitor reboxetine, and the noradrenergic and specific serotonergic antidepressant mirtazapine effectively reduced the serotonin-induced Na þ -and Ca 2 þ -currents in a dose-dependent fashion. This effect was voltage-independent and, with the exception of mirtazapine, noncompetitive. Desipramine, imipramine, trimipramine, and fluoxetine also accelerated receptor desensitization. Moclobemide and carbamazepine had no effect on the serotonin-induced cation current. By analyzing analogues of desipramine and carbamazepine, we found that a basic propylamine side chain increases the antagonistic potency of tricyclic compounds, whereas it is abolished by an uncharged carboxamide group. The antagonistic effects of antidepressants at the 5-HT 3 receptor did not correlate with their effects on membrane fluidity. In conclusion, structurally different types of antidepressants modulate the function of this ligand-gated ion channel. This may represent a yet unrecognized pharmacological principle of antidepressants.