Antipsychotic drugs antagonize human serotonin type 3 receptor currents in a noncompetitive manner.

The serotonin type 3 (5-HT(3)) receptor is the only ligand-gated ion channel receptor for serotonin (5-HT). 5-HT(3) receptors play an important role in modulating the inhibitory action of dopamine in mesocorticolimbic brain regions. Neuroleptic drugs are commonly thought to exert their psychopharmacological action mainly through dopamine and serotonin type 2 (5-HT(2)) receptors. Except for clozapine, a direct pharmacological interaction of neuroleptics with 5-HT(3) receptors has not yet been described. Using the concentration-clamp technique, we investigated the effects of flupentixol, various phenothiazines, haloperidol, clozapine and risperidone on Na(+)-inward currents through 5-HT(3) receptors stably expressed in human embryonic kidney 293 cells, and through endogenous 5-HT(3) receptors of murine N1E-115 neuroblastoma cells. In addition, we studied their effects on Ca(2+) influx, measured as a change in intracellular Ca(2+) concentrations ([Ca(2+)](i)). All neuroleptic drugs, but not risperidone, antagonized Na(+)- and Ca(2+)-inward currents evoked by 5-HT (10 microM for 2 s and 1 microM, respectively) in a voltage-independent manner. Only clozapine was a competitive antagonist, while all other compounds turned out to be noncompetitive. Fluphenazine and haloperidol affected membrane anisotropy at concentrations below their IC(50) values, indicating that a change in membrane anisotropy might contribute to their antagonistic effect at the 5-HT(3) receptor. Only structure analogues of flupentixol and fluphenazine with a lipophilic side chain were potent antagonists against 5-HT-evoked Na(+) and Ca(2+) currents. Since 5-HT(3) receptors modulate mesolimbic and mesocortical dopaminergic activity, the functional antagonism of neuroleptics at 5-HT(3) receptors may contribute to their antipsychotic efficacy and may constitute a not yet recognized pharmacological principle of these drugs.

Antidepressants are functional antagonists at the serotonin type 3 (5-HT3) receptor.

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 Ca2+, we demonstrate that different classes of antidepressants act as functional antagonists at the human 5-HT3A receptor stably expressed in HEK 293 cells and at endogenous 5-HT3 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(+)- and Ca(2)(+)-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-HT3 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.

A constitutively active pituitary adenylate cyclase activating polypeptide (PACAP) type I receptor shows enhanced photoaffinity labeling of its highly glycosylated form.

In the present study, we have analyzed a previously identified constitutively active pituitary adenylate cyclase activating polypeptide (PACAP) type I (PAC1) receptor with a deletion of the single amino acid residue Glu(261) (Y.-J. Cao, G. Gimpl, F. Fahrenholz, A mutation of second intracellular loop of pituitary adenylate cyclase activating polypeptide type I receptor confers constitutive receptor activation, FEBS Lett. 469 (2000)). This glutamic acid residue is highly conserved within the second intracellular loop of class II G protein-coupled receptors and may thus be of importance for many members of this receptor class. To explore the molecular characteristics of this mutant receptor, we performed photoaffinity labeling using previously defined photoreactive PACAP analogues. In COS cells, the PAC1 receptor was expressed in two differently glycosylated forms: a M(r) 75,000 and a M(r) 55,000 form. According to partial deglycosylation, at least three carbohydrate chains may exist in the rat PAC1 receptor expressed in COS cells. The constitutively active PAC1 receptor was expressed at the surface of COS-7 cells at the same density as the wild-type receptor. With respect to the different photoreactive PACAP analogues, the labeling specificity was the same for the wild-type versus mutant receptor: (125)I-[Lys(15)(pBz(2))]-PACAP-27 and (125)I-[Bpa(22)]-PACAP-27 were efficiently incorporated into each of the receptors, whereas (125)I-[Bpa(6)]-PACAP-27 labeled each of the receptors only to a negligible extent. This suggests that both receptors have the same or at least a very similar hormone binding site which is in close contact to Tyr(22) and Lys(15) located in the carboxy-terminal alpha-helical region of the PACAP-27 molecule. However, in comparison with the wild-type PAC1 receptor, the constitutively active receptor showed a markedly (approx. 6--8-fold) enhanced photoaffinity labeling efficiency in particular of the high glycosylated form. The enzymatically deglycosylated rat PAC1 receptor was efficiently labeled by photoreactive PACAP analogues. In contrast, nonglycosylated PAC1 receptors produced by tunicamycin treatment of the transfected COS-7 cells showed a 30-fold lower affinity for PACAP-27 and were capable of signal transduction with 30--50-fold lower potency as compared with the glycosylated PAC1 receptors.

Low cholesterol stimulates the nonamyloidogenic pathway by its effect on the alpha -secretase ADAM 10.

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 alpha-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-beta-cyclodextrin or the hydroxymethyl glutaryl-CoA reductase inhibitor lovastatin resulted in a drastic increase of secreted alpha-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 alpha-secretase activity resulted in a decreased secretion of Abeta peptides. Several mechanisms were elucidated as being the basis of enhanced alpha-secretase activity: increased membrane fluidity and impaired internalization of APP were responsible for the effect observed with methyl-beta-cyclodextrin; treatment with lovastatin resulted in higher expression of the alpha-secretase ADAM 10. Our results demonstrate that cholesterol reduction promotes the nonamyloidogenic alpha-secretase pathway and the formation of neuroprotective alpha-secretase cleaved soluble APP by several mechanisms and suggest approaches to prevention of or therapy for Alzheimer's disease.

The oxytocin receptor system: structure, function, and regulation.

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, unfortunately, the least understood. One has to conclude that the physiological regulation of the OT system will remain puzzling as long as the molecular mechanisms of genomic and nongenomic actions of steroids have not been clarified.

Regulation of receptor function by cholesterol.

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.

Oxytocin receptors and cholesterol: interaction and regulation.

Cholesterol affects the ligand binding function of the oxytocin receptor in a highly specific manner. While the structurally-related cholecystokinin receptor shows a strong correlation between the membrane fluidity and its binding function, the oxytocin receptor behaves differently. A stringent and unique requirement of the affinity state of the oxytocin receptor for structural features of the sterol molecule has been found. The molecular requirements differ both from those postulated for sterol-phospholipid interactions and from those known to be necessary for the activity of other proteins. Employing a new detergent-free subcellular fractionation protocol, a two-fold enrichment of the oxytocin receptors (10-15% of total receptors) has been detected in the cholesterol-rich, caveolin-containing membrane domains of the plasma membrane. While most of the properties of the oxytocin receptors were indistinguishable in cholesterol-poor versus cholesterol-rich membrane compartments, high-affinity oxytocin receptors localised in caveolin-enriched low-density membranes showed about a 3-fold higher stability against thermal denaturation at 37 degrees C compared with the oxytocin receptors localised in high-density membranes. Moreover, addition of cholesterol to the cholesterol-poor high-density membranes fully protected the oxytocin receptors against thermal denaturation and partially rescued high-affinity oxytocin binding. Although the membrane fluidity of the caveolin-enriched domains was lower than that in the high-density membranes, there was no correlation between the stability of oxytocin receptors and the fluidity level of the membrane domains. Finally, in a molecular modelling approach a putative cholesterol binding motif on the extracellular surface of the oxytocin receptor was found.

Human oxytocin receptors in cholesterol-rich vs. cholesterol-poor microdomains of the plasma membrane.

We analyzed the properties of a G protein-coupled receptor localized in cholesterol-poor vs. cholesterol-rich microdomains of the plasma membrane. For this purpose, the human oxytocin receptor, which is very sensitive against alterations of the membrane cholesterol level, was stably expressed in HEK293 cells. To calculate the total number of receptors independent of ligand binding studies, the oxytocin receptor was tagged with an enhanced green fluorescent protein (EGFP) which did not change the functional properties of the receptor. Only 1% of the oxytocin receptors were present in cholesterol-rich detergent-insoluble domains. In contrast, employing a detergent-free fractionation scheme that preserves the functional activity of the receptor, we detected 10-15% of the receptors in cholesterol-rich low-density membranes and therein the high-affinity state receptors were twofold enriched. In cholesterol-poor vs. cholesterol-rich domains, high-affinity oxytocin receptors behaved similar with respect to their agonist binding kinetics and GTP sensitivity. However, high-affinity oxytocin receptors localized in cholesterol-rich low-density membranes showed a markedly enhanced (t (1/2) approximately threefold) stability at 37 degrees C as compared with the oxytocin receptors localized in the cholesterol-poor high-density membranes. Addition of cholesterol to the high-density membranes fully protected the oxytocin receptors against loss of function. The importance of cholesterol to stabilize the oxytocin receptor was supported in experiments with solubilized receptors. Cholesterol markedly delayed the inactivation of oxytocin receptors solubilized with Chapso. In conclusion, the data of this report suggest that functional properties of heptahelical receptor proteins could differ in dependence of their localization in different membrane microdomains.

A mutation in the second intracellular loop of the pituitary adenylate cyclase activating polypeptide type I receptor confers constitutive receptor activation.

The pituitary adenylate cyclase activating polypeptide (PACAP) type I receptor belongs to the glucagon/secretin/vasoactive intestinal polypeptide (VIP) receptor family. We mutated and deleted an amino acid residue (E261) which is located within the second intracellular loop of the rat PACAP type I receptor and which is highly conserved among the receptor family. The wild-type receptor and the mutant receptors were efficiently expressed at the surface of COS-7 cells at nearly the same level and revealed the same high affinity for the agonist PACAP-27. The cAMP contents of COS cells transfected with the E261A, E261Q, and the deletion mutant receptor were 4.6-, 5.7-, and 6.7-fold higher as compared with COS cells transfected with the wild-type receptor. Thus, all the mutant PACAP receptors were constitutively active. The data suggest that the glutamic acid in the second intracellular loop of the PACAP receptor may be a key residue to constrain the receptor in the inactive conformation with respect to its coupling to G(s) proteins.

Non-genomic effects of progesterone on the signaling function of G protein-coupled receptors.

Progesterone at concentrations between 10 microM and 200 microM affected the calcium signaling evoked by ligand stimulation of G protein-coupled receptors expressed in several cell lines. At 160 microM progesterone the signaling of all receptors was completely abolished. The effect of progesterone was fast, reversible and was not prevented by cycloheximide indicating its non-genomic nature. Overall, the action of progesterone was more cell type-specific than receptor-specific. Our results are in contrast to a recent report [Grazzini, E., Guillon, G., Mouillac, B. and Zingg, H.H. (1998) Nature 392, 509-512] in which a direct high-affinity interaction between the oxytocin receptor and progesterone was suggested.

Cholesterol as modulator of receptor function.

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.

Photoaffinity labeling analysis of the interaction of pituitary adenylate-cyclase-activating polypeptide (PACAP) with the PACAP type I receptor.

To identify residues and domains of the peptide hormone pituitary adenylate-cyclase-activating polypeptide (PACAP) that interact with the type I receptor, two photoreactive analogues of PACAP-(1-27)-peptide were synthesized using solid-phase peptide synthesis. Phe6 or Tyr22 within the PACAP sequence were replaced by p-benzoyl-L-phenylalanine (Bz-Phe) thus creating two PACAP derivatives with a photoreactive amino acid in either the disordered N-terminal or the helical C-terminal part of the peptide. The ligand-binding properties and the efficiencies of these peptide analogues as photolabels were tested for pig brain PACAP receptors. [Bz-Phe6]-PACAP-(1-27)-peptide (Kd 1.3 nM) retained the high binding affinity of PACAP-(1-27)-peptide (Kd 0.5 nM), wheras Bz-Phe substitution of Tyr22 reduced the affinity about tenfold (Kd 4.4 nM) thus demonstrating the importance of Tyr22 for receptor binding. Monoiodination of the photoreactive analogues did not change the binding affinity of the photoreactive analogues. Photoaffinity labeling using pig brain membrane demonstrated that the 125I-labeled photoreactive analogues specifically label a 66000-Mr protein band. Photoaffinity labeling of the rat brain PACAP receptor expressed in COS cells resulted in two specifically photolabeled proteins: a major band of Mr 58000 and a minor band of Mr 78000. By treatment of photolabeled membranes with N-glycosidase F, both of the polypeptide bands were converted to a single polypeptide band of Mr 54000, which corresponds to the deglycosylated PACAP receptor. Despite its lower receptor affinity, [Bz-Phe22]-PACAP-(1-27)-peptide labeled the PACAP type I receptor in pig brain membranes and the rat receptor expressed in COS cells with much higher efficiency (20-fold for the pig receptor) than [Bz-Phe6]-PACAP-(1-27)-peptide. These findings suggest that Tyr22 in PACAP-(1-27)-peptide is located in or close to the hormone-binding site of the PACAP type I receptor. The results provide evidence that the alpha-helical C-terminal region of PACAP is directly involved in receptor binding.

Photoaffinity labeling of the human brain cholecystokinin receptor overexpressed in insect cells. Solubilization, deglycosylation and purification.

The human cholecystokinin B (CCKB) receptor was expressed in Sf9 cells by infection with recombinant baculovirus. For immunodetection a c-myc epitope tag (EQKLISEEDL) was fused at the amino-terminus of the CCKB receptor. In a second construct an additional hexa-histidine tag was introduced at the C-terminus of the CCKB receptor to enable employment of metal affinity chromatography. The two receptor constructs were expressed at densities of 6.0 +/- 1.1 pmol/mg protein and 7.2 +/- 1.1 pmol/mg protein, respectively which are 100-200-fold higher compared with the receptor amounts found in natural sources. Saturation of the binding sites with [3H]propionyl-CCK8 revealed Kd values of 4.5 +/- 0.5 nM and 7.8 +/- 0.6 nM for the CCKB receptor without or with histidine tag. In SDS/PAGE and subsequent immunodetection the histidine-tagged CCKB receptor migrated as a 55-kDa band, whereas the CCKB receptor without C-terminal modification revealed apparent molecular masses of 45 kDa and 49 kDa. The differences in the mass values observed for the two constructs suggest that the histidine tag could protect the CCKB receptor against proteolytical degradation from its C-terminus. Furthermore two new photoreactive derivatives of cholecystokinin octapeptide residues 26-33 (CCK8) with high labeling efficiency and specificity for the cholecystokinin receptor subtype B were developed: [3H]BzBz-des-Met28-[p-NH2Bz29]-CCK8 and [3H]BzBz-biotinyl-des-Met28-[p-NH2Bz29]-CCK8. Both contain the p-benzoyl-benzoyl (BzBz) residue at the N-terminus for photoactivation and a p-aminobenzoyl (p-NH2BZ) residue instead of Met28-Gly29 in cholecystokinin. Enzymatic deglycosylation of the CCKB receptor with N-glycosidase F after photoaffinity labeling demonstrated that the CCKB receptor with three potential glycosylation sites was slightly glycosylated, amounting to a molecular mass of about 4 kDa. Using the biotinylated cholecystokinin derivative the photoaffinity-labeled CCKB receptor could be purified 1260-fold by a two-step procedure including affinity chromatography on a streptavidin/avidin agarose matrix. For purification of the native receptor, an improved solubilization protocol for the CCKB receptor using dodecyl beta-D-maltopyranoside was developed. The solubilized CCKB receptors with C-terminal histidine tag retained their ligand binding characteristics after chromatography on a nickel affinity matrix.

Alteration of the myometrial plasma membrane cholesterol content with beta-cyclodextrin modulates the binding affinity of the oxytocin receptor.

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)

Expression of the human oxytocin receptor in baculovirus-infected insect cells: high-affinity binding is induced by a cholesterol-cyclodextrin complex.

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)

The amino-terminal fragment of the adenylate cyclase activating polypeptide (PACAP) receptor functions as a high affinity PACAP binding domain.

The PACAP receptor represents a member of a novel subfamily of G-protein coupled receptors with a common structurally conserved extracellular domain of about 150 amino acids. We have addressed the question whether this extracellular amino-terminus of the PACAP type I receptor can solely function as a PACAP binding domain. For that purpose a cDNA was constructed that encodes the membrane-anchored amino-terminus of the rat PACAP receptor including the decapeptide epitope EQKLISEEDL for immunodetection. COS-7 cells were transfected with this cDNA and a comparable construct of the wild-type receptor. Binding analysis showed that the amino-terminal fragment of the PACAP receptor bound PACAP with high-affinity (Kd = 3.8 nM; Bmax = 12.8 pmol/mg protein). In comparison to the full-length receptor (Kd = 0.2 nM; Bmax = 1.96 pmol/mg protein) its affinity was reduced by a factor of about 20. The results suggest that the amino-terminus of the PACAP receptor functions as the major binding site for its ligand.

Modulation of potassium currents in cultured murine microglial cells by receptor activation and intracellular pathways.

The electrophysiological properties of ameboid microglia from rodent brain are dominated by inwardly rectifying potassium channels and by the lack of outward currents. This channel pattern results in a distinct physiological behavior: depolarizing events, e.g. following adenosine triphosphate receptor activation, can lead to a long lasting membrane depolarization. Here we address the question whether this resting K+ channel activity can be modulated. Intracellular application of guanosine 5'-O-(3-thiotriphosphate) induced an outward current and led to a complete disappearance of the inward current inward rectifier potassium current as measured with the patch clamp technique. Moreover, an elevation in cytosolic calcium concentration (to 1.6 microM) via intracellular perfusion reversibly blocked the inward current. The inhibition of inward currents by guanosine 5'-O-(3-thiotriphosphate) could be enhanced by additional adenosine triphosphate receptor activation. Adenosine triphosphate or tumor necrosis factor receptor activation alone could lead to a transient partial block of the inward rectifier and to the transient appearance of a delayed outward current. We conclude that the activity of the microglia K+ channels and thus the physiological behavior of microglia can be modulated on a time scale of seconds by receptor activation and distinct intracellular pathways.

Conversion of the myometrial oxytocin receptor from low to high affinity state by cholesterol.

Reconstitution experiments with the myometrial oxytocin receptor from guinea pig showed a strong requirement of the oxytocin receptor binding function for the presence of cholesterol in preformed liposomes. To investigate the effect of cholesterol on the oxytocin receptor in the plasma membrane, a new method to modify the membrane cholesterol content was developed. With substituted cyclodextrins we were able to selectively deplete the myometrial plasma membrane of cholesterol. Vice versa, incubation of cholesterol-depleted membranes with a soluble cholesterol cyclodextrin complex restored the cholesterol content of the plasma membrane. Binding experiments showed, that with the removal of cholesterol from the membrane, the oxytocin receptor was converted from high to low affinity state. Increasing the cholesterol content of the membrane again restored the high binding affinity. Substitution of membrane cholesterol with other steroids showed a strong dependence of the oxytocin receptor function on the structure of the cholesterol molecule. Experiments with the detergent solubilized oxytocin receptor and with oxytocin receptors expressed in the Baculovirus/Sf9 cell system provided further evidence for a direct interaction of the oxytocin receptor with cholesterol.

Molecular structure analysis of the pituitary adenylate cyclase activating polypeptide type I receptor from pig brain.

In pig brain a type I receptor for pituitary adenylate cyclase activating polypeptide (PACAP) has been identified and structurally characterized. Scatchard analysis of the equilibrium binding data indicates a single class of binding sites with Kd of 0.50 nM and Bmax of 2.52 pmol/mg. The receptors could be efficiently solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1- propanesulfonate (Chapso) without altering their ligand binding parameters (Kd = 0.60 nM; Bmax = 2.46 pmol/mg). In both preparations the ligand-receptor complex was identified as a 68 kDa polypeptide by affinity crosslinking with 125I-PACAP-27 and disuccinimidyl suberate. Employment of nonreducing conditions decreased the electrophoretic mobility of the complex to a 60 kDa species suggesting that the native PACAP receptor exists in a compact conformation stabilized by intramolecular disulfide bridges. Disulfide bonds are also important for the ligand binding activity of the PACAP receptor since pretreatment of the membranes with dithiothreitol led to complete inhibition of PACAP binding and to 61% dissociation of the ligand-receptor complex. Among different sulfhydryl selective reagents tested, p-chloromercuriphenylsulfonic acid was found to inhibit PACAP binding to its receptor in a dose-dependent fashion (IC50 = 0.5 mM) suggesting that one or more sulfhydryl groups are located close to the ligand binding domain of the receptor. Desialylation of the affinity-labeled PACAP receptor with neuraminidase revealed a 61 kDa protein, whereas deglycosylation with N-glycosidase F decreased the M(r) to 52,000. Chromatography on a series of lectin agaroses showed the highest affinity for wheat germ agglutinin (WGA). WGA was also effective in dose-dependent inhibition of PACAP binding activity. Our results characterize the pig PACAP receptor as a tri- or tetraantennary non- or low fucosylated complex type glycoprotein with a protein core of about 49 kDa containing several terminal sialyl residues.

Extracellular ATP-induced currents in astrocytes: involvement of a cation channel.

Whole-cell currents were measured with the perforated patch clamp technique in cultured rat astrocytes to analyze the underlying ionic mechanism for a P2-purinoceptor-mediated depolarization. ATP (100 microM) induced an inward current with a mean amplitude of 130 pA and an EC50 of 17 microM. The response desensitized during a 1 min application. Replacement of extracellular Na+ with NMDG or K+ abolished the ATP-evoked inward current. Replacement of Na+ with choline, however, resulted in an ATP-evoked response of one-third the amplitude in normal solution. This is indicative of a cation rather than Na+ channel. However, due to difficulties in voltage-clamping these gap junction-coupled cells at voltages different from the membrane resting potential, the current reversal potential could not be determined. Measurements with K(+)-sensitive microelectrodes showed that 100 microM ATP lowered the intracellular K+ concentration. Replacement of extracellular Ca2+ or Cl- did not alter the ATP-induced inward currents. Fura-2 imaging experiments revealed a transient rise of the intracellular Ca2+ concentration during ATP application. Removal of extracellular Ca2+ did not influence the peak response; it did, however, shorten the time course. These results and previous observations that the permeability changes are caused by a P2x receptor are indicative of an ATP-sensitive cation conductance. In addition, cytoplasmic Ca2+ is increased by mobilization from intracellular stores, and by additional influx across the cell membrane. Extracellular ATP released by neurons could evoke K+ release from astrocytes as well as be a mediator for cation changes that signal cell activation processes when released by damaged cells.