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 plasma membrane V-ATPase of Manduca sexta larval midgut is an electrogenic proton pump located in goblet cell apical membranes (GCAM); it energizes, by the voltage component of its proton motive force, an electrophoretic K+/nH+ antiport and thus K+ secretion (Wieczorek, H., Putzenlechner, M., Zeiske, W., and Klein, U. (1991) J. Biol Chem. 266, 15340-15347). Midgut transepithelial voltage, indicating net active K+ transport, was found to be more than 100 mV during intermoult stages but was abolished during moulting. Simultaneously, ATP hydrolysis and ATP-dependent proton transport in GCAM vesicles were found to be reduced to 10-15% of the intermoult level. Immunocytochemistry of midgut cryosections as well as SDS-polyacrylamide gel electrophoresis and immunoblots of GCAM demonstrated that loss of ATPase activity paralleled the disappearance of specific subunits. The subunits missing were those considered to compose the peripheral V1 sector, whereas the membrane integral V0 subunits remained in the GCAM of moulting larvae. The results provide, for the first time, evidence that a V-ATPase activity can be controlled in vivo by the loss of the peripheral V1 domain.

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)

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Angiotensin receptor blockers (ARBs) are well-tolerated drugs that are known to be useful for inhibiting activity of the reninangiotensin (RAS) system, treating hypertension and reducing the risk for cardiovascular disease. However, inhibition of the RAS does not control all pathophysiological mechanisms of hypertension or cardiovascular risk and many patients continue to suffer from cardiovascular events and metabolic disturbances despite being treated with an ARB, an angiotensin-converting enzyme inhibitor or both, in addition to other standard therapies for cardiovascular disease. Recently, it has become apparent that bifunctional molecules can be designed that do more than just block AT 1 receptors and that can target additional mechanisms of hypertension, cardiovascular disease and diabetes besides just increased activity of the renin-angiotensin system. Specifically, next generation ARBs are becoming available that are intended to not only antagonize AT 1 receptors, but also block endothelin receptors, function as nitric oxide donors, inhibit neprilysin activity and increase natriuretic peptide levels, or stimulate the peroxisome proliferator-activated receptor c (PPARc). In this review, we: (1) discuss the potential importance of multifunctional ARBs that can reduce cardiovascular and metabolic risk through multiple mechanisms that go beyond just inhibition of the renin-angiotensin system and (2) describe specific examples of next generation ARBs in development that are intended to do more than simply block AT 1 receptors.

Dual inhibition of angiotensin-converting enzyme (ACE) and neprilysin (NEP) by drugs such as omapatrilat produces superior antihypertensive efficacy but cause high incidence of angioedema. We examined whether dual inhibition of angiotensin AT1 receptor (ARB) and NEP (ARB-NEPI, valsartan-candoxatril) provides similar efficacy to omapatrilat without the risk of angioedema. Activity of test compounds at the targets was assayed using fluorescence-based enzyme assays (ACE, NEP, aminopeptidase P) or competition binding assays (AT1). Target engagement in vivo (ACE, AT1, and NEP) was quantified by measuring inhibition of angiotensin-pressor responses and potentiation of atrial natriuretic peptide-induced urinary cyclic guanosine monophosphate (cGMP) output in rats. Tracheal plasma extravasation (TPE) was used as a surrogate to assess propensity of compounds to promote upper airway angioedema. Antihypertensive efficacy in renin-dependent and -independent states was measured in spontaneously hypertensive rats and deoxycorticosterone acetate salt hypertensive rats, respectively. Administration of omapatrilat and coadministration of valsartan and candoxatril blocked angiotensin induced vasopressor responses and potentiated atrial natriuretic peptide-induced increase in urinary cGMP output. In spontaneously hypertensive rats, valsartan, omapatrilat, and valsartan-candoxatril combination all produced reduction in blood pressure to a similar extent, whereas candoxatril was ineffective. In deoxycorticosterone acetate rats, omapatrilat, candoxatril, and valsartan-candoxatril combination but not valsartan produced reduction in blood pressure. Antihypertensive doses of omapatrilat produced robust increases in TPE; by contrast, valsartan, candoxatril, or their combination did not increase TPE. Pretreatment with icatibant, a bradykinin B2 antagonist, abolished omapatrilat-induced TPE but not its antihypertensive effects. On the background of NEP inhibition, suppression of the renin-angiotensin system through ARB and ACE inhibition shows a similar antihypertensive efficacy but exerts differential effects on bradykinin metabolism and TPE indicative of reduced risk of angioedema. Thus, dual AT1 receptor blockade and NEP inhibition is potentially an attractive approach to retain the excellent antihypertensive effects of omapatrilat but with a superior safety profile.

The ␣-subunit of eukaryotic initiation factor 2B (eIF-2B), a guanine nucleotide exchange protein that functions in regulation of translation, was observed to associate with the carboxyl-terminal cytoplasmic domains of the ␣ 2A -and ␣ 2B -adrenergic receptors in a yeast twohybrid screen of a cDNA library prepared from 293 cells. This protein association was confirmed in vitro by affinity chromatography and was shown to be specific for a subset of G protein-coupled receptors, including the ␣ 2A -, ␣ 2B -, ␣ 2C -, and ␤ 2 -adrenergic receptors, but not the vasopressin (V 2 ) receptor. Association of these proteins in vivo was confirmed by specific co-immunoprecipitation of eIF-2B␣ with full-length ␤ 2 -adrenergic receptors expressed in transfected 293 cells and by fluorescence microscopy showing co-localization of these proteins in intact cells. Remarkably, eIF-2B␣ co-localized with receptors exclusively in regions of the plasma membrane that are in contact with the extracellular medium, but failed to associate with membranes making cell-cell contacts. Overexpression of eIF-2B␣ in 293 cells caused a small (ϳ15%) but significant enhancement of ␤ 2 -adrenergic receptor-mediated activation of adenylyl cyclase, without affecting forskolin or V 2 receptor-mediated activation. These observations suggest a new role for a previously identified guanine nucleotide exchange protein in membrane biology and cell signaling.G protein-coupled receptors interact with several classes of cytoplasmic proteins, including heterotrimeric G proteins, kinases, phosphatases, and arrestins (1-4). Specific roles of these protein associations in receptor signaling and regulation are now well established. These protein interactions were first inferred from their functional effects on receptor signaling and desensitization before direct physical associations of these proteins with receptors were observed biochemically (5-8). This observation raises the possibility that receptors may interact with additional cellular proteins that could play unanticipated roles in determining the efficacy or specificity of receptor-G protein coupling.We have investigated this possibility by searching for novel protein interactions with adrenergic receptors, focusing on the carboxyl-terminal cytoplasmic domain because mutations within this domain have pleiotropic effects on receptor physiology (9 -12). 1 Using interaction cloning and biochemical techniques, we have observed that several subtypes of adrenergic receptors associate specifically with eIF-2B␣, 2 the smallest subunit of a cytoplasmic guanine nucleotide exchange factor. While this protein has a well defined role in regulation of translation, it has never been shown previously to interact with any membrane receptor. Immunocytochemical studies suggest that receptors associate with eIF-2B␣ only in restricted regions of the plasma membrane, and functional studies suggest that this protein interaction may play a role in the regulation of receptor-mediated signaling. EXPERIMENTAL PROCEDURESYeast Two-hybrid Clo...