Stimulated Inositol Phosphate Research Articles

Thyrotropin Stimulates the Generation of Inositol 1,4,5-Trisphosphate in Human Thyroid Cells

Dual activation by TSH of the phospholipase C and cAMP cascades has been reported in human thyroid cells. In contrast, Singh et al. reported convincing data in FRTL-5 thyrocytes arguing against such an effect in this model. Their data in FRTL-5 cells indicated no increase in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in response to TSH. Therefore, the authors questioned results previously obtained on human cells by cruder methodology. We investigated the formation of inositol phosphates by HPLC techniques in human thyroid slices to separate the inositol phosphate isomers. Ins(1,4,5)P3, inositol 1,3,4-trisphosphate, and inositol 1,3,4,5-tetrakisphosphate were increased after TSH stimulation. The effect of TSH in human thyroid cells was reproduced by recombinant TSH and prevented by antibodies blocking the TSH receptor. Thyroid-stimulating antibodies at concentrations eliciting a cAMP response equivalent to TSH failed to stimulate inositol phosphate generation. TSH, but not thyroid-stimulating antibodies, activates both cAMP and the phospholipase C cascade in human thyroid as now demonstrated by an increase in Ins(1,4,5)P3 and its inositol phosphate metabolites. Therefore, this effect cannot be extrapolated to the FRTL-5 cell line. The apparent discrepancy may be due to a difference between species (human vs. rat) or to the loss of the fresh tissue properties in a cell line. The dual effect of TSH in human cells, through cAMP on secretion of thyroid hormones and through the diacylglycerol, Ins(1,4,5)P3 Ca2+ pathway on thyroid hormone synthesis, implies the possible separation of these effects in thyroid disease.

Inhibition of Serotonin 5-Hydroxytryptamine2C Receptor Function through Heterodimerization: RECEPTOR DIMERS BIND TWO MOLECULES OF LIGAND AND ONE G-PROTEIN

Although dimerization appears to be a common property of G-protein-coupled receptors (GPCRs), it remains unclear whether a GPCR dimer binds one or two molecules of ligand and whether ligand binding results in activation of one or two G-proteins when measured using functional assays in intact living cells. Previously, we demonstrated that serotonin 5-hydroxytryptamine2C (5-HT2C) receptors form homodimers (Herrick-Davis, K., Grinde, E., and Mazurkiewicz, J. (2004) Biochemistry 43, 13963-13971). In the present study, an inactive 5-HT(2C) receptor was created and coexpressed with wild-type 5-HT2C receptors to determine whether dimerization regulates receptor function and to determine the ligand/dimer/G-protein stoichiometry in living cells. Mutagenesis of Ser138 to Arg (S138R) produced a 5-HT2C receptor incapable of binding ligand or stimulating inositol phosphate (IP) signaling. Confocal fluorescence imaging revealed plasma membrane expression of yellow fluorescent protein-tagged S138R receptors. Expression of wild-type 5-HT2C receptors in an S138R-expressing stable cell line had no effect on ligand binding to wild-type 5-HT2C receptors, but inhibited basal and 5-HT-stimulated IP signaling as well as constitutive and 5-HT-stimulated endocytosis of wild-type 5-HT2C receptors. M1 muscarinic receptor activation of IP production was normal in the S138R-expressing cells. Heterodimerization of S138R with wild-type 5-HT2C receptors was visualized in living cells using confocal fluorescence resonance energy transfer (FRET). FRET was dependent on the donor/acceptor ratio and independent of the receptor expression level. Therefore, inactive 5-HT2C receptors inhibit wild-type 5-HT2C receptor function by forming nonfunctional heterodimers expressed on the plasma membrane. These results are consistent with a model in which one GPCR dimer binds two molecules of ligand and one G-protein and indicate that dimerization is essential for 5-HT receptor function.

Probing the Molecular Mechanism of Interaction between 4-n-Butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine (AC-42) and the Muscarinic M1Receptor: Direct Pharmacological Evidence That AC-42 Is an Allosteric Agonist

4-n-Butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine hydrogen chloride (AC-42) is a selective agonist of the muscarinic M(1) receptor previously suggested to interact with an "ectopic" site on this receptor. However, the pharmacological properties of this site (i.e., whether it overlaps to any extent with the classic orthosteric site or represents a novel allosteric site) remain undetermined. In the present study, atropine or pirenzepine significantly inhibited the ability of either carbachol or AC-42 to stimulate inositol phosphate accumulation or intracellular calcium mobilization in Chinese hamster ovary (CHO) cells stably expressing the human M(1) receptor. However, the interaction between either of these antagonists and AC-42 was characterized by Schild slopes significantly less than unity. Increasing the concentrations of atropine revealed that the Schild regression was curvilinear, consistent with a negative allosteric interaction. More direct evidence for an allosteric mode of action of AC-42 was obtained in [(3)H]N-methylscopolamine ([(3)H]NMS) binding studies, in that both AC-42 and the prototypical modulator gallamine failed to fully inhibit specific [(3)H]NMS binding in a manner that was quantitatively described by an allosteric model applied to both modulator data sets. Furthermore, AC-42 and gallamine significantly retarded the rate of [(3)H]NMS dissociation from CHO-hM(1) cell membranes, conclusively demonstrating their ability to bind to a topographically distinct site to change M(1) receptor conformation. These data provide the first direct evidence that AC-42 is an allosteric agonist that activates M(1) receptors in the absence of the orthosteric agonist.

Prostaglandin I2 Production and cAMP Accumulation in Response to Acidic Extracellular pH through OGR1 in Human Aortic Smooth Muscle Cells

Ovarian cancer G-protein-coupled receptor 1 (OGR1) and GPR4 have recently been identified as proton-sensing or extracellular pH-responsive G-protein-coupled receptors stimulating inositol phosphate production and cAMP accumulation, respectively. In the present study, we found that OGR1 and GPR4 mRNAs were expressed in human aortic smooth muscle cells (AoSMCs). Acidic extracellular pH induced inositol phosphate production, a transient increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), and cAMP accumulation in these cells. When small interfering RNAs (siRNAs) targeted for OGR1 and GPR4 were transfected to the cells, the acid-induced inositol phosphate production and [Ca(2+)](i) increase were markedly inhibited by the OGR1 siRNA but not by the GPR4 siRNA. Unexpectedly, the acid-induced cAMP accumulation was also largely inhibited by OGR1 siRNA but only slightly by GPR4 siRNA. Acidic extracellular pH also stimulated prostaglandin I2 (PGI(2)) production, which was again inhibited by OGR1 siRNA. The specific inhibitors for extracellular signal-regulated kinase kinase and cyclooxygenase attenuated the acid-induced PGI(2) production and cAMP accumulation without changes in the inositol phosphate production. A specific inhibitor of phospholipase C also inhibited the acid-induced cAMP accumulation. In conclusion, OGR1 is a major receptor involved in the extracellular acid-induced stimulation of PGI(2) production and cAMP accumulation in AoSMCs. The cAMP accumulation may occur through OGR1-mediated stimulation of the phospholipase C/cyclooxygenase/PGI(2) pathway.

Gonadotropin-Releasing Hormone II Stimulates Female Sexual Behavior in Marmoset Monkeys

GnRH II (pGlu-His-Trp-Ser-Try-Gly-Leu-Arg-Pro-GlyNH2), an evolutionarily conserved member of the GnRH family, stimulates reproductive behavior in a number of vertebrates. To explore a role for GnRH II in regulating primate sexual behavior, eight adult female common marmosets, each fitted with an indwelling intracerebroventricular (icv) cannula, were ovariectomized, implanted subcutaneously with empty (n = 4) or estradiol-filled (n = 4) SILASTIC brand capsules, and pair housed with an adult male mate. After icv infusion of vehicle or peptides, females were placed in an observation cage for 90 min, out of visual contact with other marmosets, before the 30-min behavioral test with their male partner. Compared with vehicle, GnRH II (1 and 10 microg) increased the total number of proceptive (sexual solicitation) behaviors (tongue flicking, proceptive stares, and frozen postures) exhibited by females toward their pair mates and specifically increased the frequency of freeze postures. Effects were maximal at 1 microg and not dependent upon estradiol supplementation. GnRH II agonists/GnRH I antagonists 135-18 (1 microg) and 132-25 (1 microg), which stimulate inositol phosphate production via the marmoset type II receptor, increased the frequency of total proceptive behavior but did not specifically stimulate freeze-posture behavior. In contrast, GnRH I, at 1 mug, did not alter the frequency of proceptive behaviors. Female receptivity (female compliance with male sexual behavior) was not altered by any of the peptides tested. These findings implicate a role for GnRH II and the cognate GnRH type II receptor in stimulating female marmoset sexual behavior.

Phosphorylation-independent Regulation of Metabotropic Glutamate Receptor 1 Signaling Requires G Protein-coupled Receptor Kinase 2 Binding to the Second Intracellular Loop

Metabotropic glutamate receptors (mGluRs) are members of a unique class of G protein-coupled receptors (class III) that include the calcium-sensing and gamma-aminobutyric acid type B receptors. The activity of mGluRs is regulated by second messenger-dependent protein kinases and G protein-coupled receptor kinases (GRKs). The attenuation of both mGluR1a and mGluR1b signaling by GRK2 is phosphorylation- and beta-arrestin-independent and requires the concomitant association of GRK2 with both the receptor and Galpha(q/11). G protein interactions are mediated, in part, by the mGluR1 intracellular second loop, but the domains required for GRK2 binding are unknown. In the present study, we showed that GRK2 binds to the second intracellular loop of mGluR1a and mGluR1b and also to the mGluR1a carboxyl-terminal tail. Alanine scanning mutagenesis revealed a discrete domain within loop 2 that contributes to GRK2 binding, and the mutation of either lysine 691 or 692 to an alanine within this domain resulted in a loss of GRK2 binding to both mGluR1a and mGluR1b. Mutation of either Lys(691) or Lys(692) prevented GRK2-mediated attenuation of mGluR1b signaling, whereas the mutation of only Lys(692) prevented GRK2-mediated inhibition of mGluR1a signaling. Thus, the mGluR1a carboxyl-terminal tail may also be involved in regulating the signaling of the mGluR1a splice variant. Taken together, our findings indicated that kinase binding to an mGluR1 domain involved in G protein-coupling is essential for the phosphorylation-independent attenuation of signaling by GRK2.

N-Acetylcysteine Decreases Angiotensin II Receptor Binding in Vascular Smooth Muscle Cells

Antioxidants seem to inhibit angiotensin II (Ang II) actions by consuming stimulated reactive oxygen species. An alternative hypothesis was investigated: Antioxidants that are also strong reducers of disulfide bonds inhibit the binding of Ang II to its surface receptors with consequent attenuation of signal transduction and cell action. Incubation of cultured vascular smooth muscle cells, which possess Ang II type 1a receptors, with the reducing agent n-acetylcysteine (NAC) for 1 h at 37 degrees C resulted in decreased Ang II radioligand binding in a concentration-dependent pattern. NAC removal restored Ang II binding within 30 min. Incubation with n-acetylserine, a nonreducing analogue of NAC, did not lower Ang II binding, and oxidized NAC was less effective than reduced NAC in lowering Ang II binding. NAC did not decrease Ang II type 1a receptor protein content. Other antioxidants regulated Ang II receptors differently: alpha-Lipoic acid lowered Ang II binding after 24 h, and vitamin E did not lower Ang II binding at all. NAC inhibited Ang II binding in cell membranes at 21 or 37 but not 4 degrees C. Dihydrolipoic acid (the reduced form of alpha-lipoic acid), which contains free sulfhydryl groups as NAC does, decreased Ang II receptor binding in cell membranes, whereas alpha-lipoic acid, which does not contain free sulfhydryl groups, did not. Ang II-stimulated inositol phosphate formation was decreased by preincubation with NAC (1 h) or alpha-lipoic acid (24 h) but not vitamin E. In conclusion, certain antioxidants that are reducing agents lower Ang II receptor binding, and Ang II-stimulated signal transduction is decreased in proportion to decreased receptor binding.

Lysophosphatidylserine stimulates leukemic cells but not normal leukocytes

In this study, we observed that lysophosphatidylserine (LPS) stimulated intracellular calcium ([Ca 2+] i) increase in leukemic cells but not in normal human peripheral blood mononuclear cells. LPS also stimulated [Ca 2+] i increase in human leukemic THP-1 cells. LPS-stimulated [Ca 2+] i increase was inhibited by U-73122 but not by U-73343. LPS also stimulated inositol phosphates formation in THP-1 cells, suggesting that LPS stimulates calcium signaling via phospholipase C activation. Moreover, pertussis toxin (PTX) completely inhibited [Ca 2+] i increase by LPS, indicating the activation of PTX-sensitive G-proteins. We also found that LPS-induced [Ca 2+] i increase was completely inhibited by suramin, suggesting G-protein coupled receptor activation. Since LPS specifically stimulates PTX-sensitive G-proteins, phospholipase C-dependent [Ca 2+] i increase in leukemic cells but not normal peripheral blood leukocytes, LPS receptor may be associated with leukemia.

Potentiation of bradykinin actions by analogues of the bradykinin potentiating nonapeptide BPP 9α

Synthetic analogues of the bradykinin potentiating nonapeptide BPP 9α indicate significantly different structural requirements for potentiation of the bradykinin (BK)-induced smooth muscle contraction (GPI) and the inhibition of isolated somatic angiotensin I-converting enzyme (ACE). The results disprove the ACE inhibition as the only single mechanism and also the direct interaction of potentiating peptides with the bradykinin receptors in transfected COS-7 cells as molecular mechanism of potentiation. Our results indicate a stimulation of inositol phosphates (IP n ) formation independently from the B 2 receptor. Furthermore, the results with La 3+ support the role of extracellular Ca 2+ and its influx through corresponding channels. The missing effect of calyculin on the GPI disproves the role of phosphatases in the potentiating action. These experimental studies should not only contribute to a better understanding of the potentiating mechanisms but also incorporate a shift in the research towards the immune system, in particular towards the immunocompetent polymorphonuclear leukocytes. The chemotaxis of these cells can be potentiated most likely by exclusive inhibition of the enzymatic degradation of bradykinin. Thus the obtained results give evidence that the potentiation of the bradykinin action can occur by different mechanisms, depending on the system and on the applied potentiating factor.

Addition of a signal peptide sequence to the α1D‐adrenoceptor gene increases the density of receptors, as determined by [3H]‐prazosin binding in the membranes

1. Both in mammalian tissues and in transfected cells, only low levels of alpha1D-adrenoceptors are detected in radioligand binding studies. It has been implicated that the comparatively long N-terminal tail of the alpha1D-adrenoceptor is responsible for the inefficient surface expression of the receptor. 2. In the present study, we created gene constructs for six N-terminally truncated variants of the human alpha1D-adrenoceptor. These constructs were used to transfect Neuro2A cells. We show that the density of alpha1D-adrenoceptors, observed by [3H]-prazosin binding, gradually increased with longer truncations of the N-terminus. This seems to indicate that the long N-terminal tail nonspecifically interferes with receptor translocation to the plasma membrane. 3. The addition of a 16 amino acids long signal peptide to the N-terminus of the wild-type alpha1D-adrenoceptor increased the density of receptor binding sites 10-fold in Neuro2A and COS-7 cells. This indicates that, after the addition of a signal peptide, the long N-terminal tail of the alpha1D-adrenoceptor does not interfere with proper translocation of the receptor to the plasma membrane. This, in turn, indicates that the N-terminal tail of the wild-type alpha1D-adrenoceptor, merely by its long length, hinders the first transmembrane helix of the receptor from being a signal anchor. 4. Neither the wild-type alpha1D-adrenoceptor (for which the expression level of [3H]-prazosin binding sites is low) nor the truncated alpha1D-adrenoceptor variant (for which the expression level of [3H]-prazosin binding sites is high) showed any constitutive activity in stimulating inositol phosphate accumulation. This indicates that the low expression level of [3H]-prazosin binding sites, after transfection with the wild-type alpha1D-adrenoceptor, is not caused by constitutive activity of the receptor and subsequent receptor downregulation.

Sphingosylphosphorylcholine Antagonizes Proton-Sensing Ovarian Cancer G-Protein-Coupled Receptor 1 (OGR1)-Mediated Inositol Phosphate Production and cAMP Accumulation

Ovarian cancer G-protein-coupled receptor 1 (OGR1), previously proposed as a receptor for sphingosylphosphorylcholine (SPC), has recently been identified as a proton-sensing or extracellular pH-responsive G-protein-coupled receptor stimulating inositol phosphate production, reflecting the activation of phospholipase C. In the present study, we found that acidic pH stimulated cAMP accumulation, reflecting the activation of adenylyl cyclase, in addition to inositol phosphate production in OGR1-expressing cells. The cAMP response was hardly affected by the inhibition of phospholipase C. SPC inhibited the acidification-induced actions in a pH-dependent manner, while no OGR1-dependent agonistic action of SPC was observed. Thus, the dose-response curves of the proton-induced actions were shifted to the right in the presence of SPC regardless of stereoisoform. The antagonistic property was also observed for psychosine and glucosylsphingosine. In conclusion, OGR1 stimulation may lead to the activation of adenylyl cyclase in addition to phospholipase C in response to extracellular acidification but not to SPC. However, SPC and related lysolipids antagonize the proton-induced and OGR1-mediated actions.

TDAG8 Is a Proton-sensing and Psychosine-sensitive G-protein-coupled Receptor

T cell death-associated gene 8 (TDAG8) has been reported to be a receptor for psychosine. Ovarian cancer G-protein-coupled receptor 1 (OGR1) and GPR4, G-protein-coupled receptors (GPCRs) closely related to TDAG8, however, have recently been identified as proton-sensing or extracellular pH-responsive GPCRs that stimulate inositol phosphate and cAMP production, respectively. In the present study, we examined whether TDAG8 senses extracellular pH change. In the several cell types that were transfected with TDAG8 cDNA, cAMP was markedly accumulated in response to neutral to acidic extracellular pH, with a peak response at approximately pH 7.0-6.5. The pH effect was inhibited by copper ions and was reduced or lost in cells expressing mutated TDAG8 in which histidine residues were changed to phenylalanine. In the membrane fractions prepared from TDAG8-transfected cells, guanosine 5'-O-(3-thiotriphosphate) binding activity and adenylyl cyclase activity were remarkably stimulated in response to neutral and acidic pH. The concentration-dependent effect of extracellular protons on cAMP accumulation was shifted to the right in the presence of psychosine. The inhibitory psychosine effect was also observed for pH-dependent actions in OGR1- and GPR4-expressing cells but not for prostaglandin E(2)- and sphingosine 1-phosphate-induced actions in any pH in native and sphingosine 1-phosphate receptor-expressing cells. Glucosylsphingosine and sphingosylphosphorylcholine similarly inhibited the pH-dependent action, although to a lesser extent. Psychosine-sensitive and pH-dependent cAMP accumulation was also observed in mouse thymocytes. We concluded that TDAG8 is one of the proton-sensing GPCRs coupling to adenylyl cyclase and psychosine, and its related lysosphingolipids behave as if they were antagonists against protein-sensing receptors, including TDAG8, GPR4, and OGR1.

Discovery of the first non-peptide full agonists for the human bradykinin B(2) receptor incorporating 4-(2-picolyloxy)quinoline and 1-(2-picolyl)benzimidazole frameworks.

In the course of our studies on non-peptide bradykinin (BK) B(2) receptor ligands, it was suggested that the 4-substituent of the quinoline ring may play a critical role in determining binding affinities for human and guinea pig B(2) receptors, as well as agonist/antagonist properties. We carried out an extensive investigation to elucidate the structure-activity relationships (SAR) for this key pharmacophore. Introduction of lower alkoxy groups to the 4-position of the quinoline ring of 3 led to the identification of 4-ethoxy derivative 22b as a unique partial agonist. This compound significantly stimulated inositol phosphates (IPs) formation in Chinese hamster ovary cells expressing the cloned human B(2) receptor at concentrations greater than 10 nM and displayed one-tenth of the intrinsic activity of BK. The agonist activity of 22b was selective for the B(2) receptor and was inhibited by selective peptide and non-peptide B(2) antagonists. On the other hand, 22b strongly suppressed BK-induced IPs formation through the cloned human B(2) receptor. Further studies on the key pharmacophore led to identification of a 2-picolyloxy moiety as a powerful agonist switch, leading to the discovery of a potent and efficacious non-peptide B(2) agonist, 19a. Successive optimization of the acyl side chain afforded 38, which exhibited full agonist activity on stimulation of IPs formation. Furthermore, this strategy could be applied successfully to the benzimidazole series. The representative 1-(2-picolyl)benzimidazole derivative 47c increased PGE(2) production at a 1 microM concentration to the same level as the maximum effect of BK. Thus, we have established the medicinal chemistry modifications required to convert our highly potent non-peptide B(2) antagonists to agonists with potent efficacy.

A Novel Effect of Bismuth Ions: SELECTIVE INHIBITION OF THE BIOLOGICAL ACTIVITY OF GLYCINE-EXTENDED GASTRIN

Although bismuth salts have been used for over two centuries for the treatment of various gastrointestinal disorders, the mechanism of their therapeutic action remains controversial. Because gastrins bind two trivalent ferric ions with high affinity, and because ferric ions are essential for the biological activity of glycine-extended gastrin 17, we have investigated the hypothesis that trivalent bismuth ions influence the biological activity of gastrins. Binding of bismuth ions to gastrins was measured by fluorescence quenching and NMR spectroscopy. The effects of bismuth ions on gastrin-stimulated biological activities were measured in inositol phosphate, cell proliferation, and cell migration assays. Fluorescence quenching experiments indicated that both glycine-extended and amidated gastrin 17 bound two bismuth ions. The NMR spectral changes observed on addition of bismuth ions revealed that Glu-7 acted as a ligand at the first bismuth ion binding site. In the presence of bismuth ions the ability of glycine-extended gastrin 17 to stimulate inositol phosphate production, cell proliferation, and cell migration was markedly reduced. In contrast, bismuth ions had little effect on the affinity of the CCK-2 receptor for amidated gastrin 17, or on the stimulation of inositol phosphate production by amidated gastrin 17. We conclude that bismuth ions may act, at least in part, by blocking the effects of glycine-extended gastrin 17 on cell proliferation and cell migration in the gastrointestinal tract. This is the first report of a specific inhibitory effect of bismuth ions on the action of a gastrointestinal hormone.

Identification and characterization of a reptilian GnRH receptor from the leopard gecko

Gonadotropin-releasing hormone (GnRH) plays a pivotal role in the regulation of reproductive functions through interactions with its specific receptor. We describe the first molecular cloning and characterization of a full-length GnRH receptor (GnRHR) from the leopard gecko Eublepharis macularius. It has a distinct genomic structure consisting of five exons and four introns, compared with all the other reported GnRHR genes. A native GnRH form, cGnRH-II, stimulated inositol phosphate (IP) production in COS-7 cells transiently transfected with the GnRHR, in a dose dependent manner. The mRNA was expressed in all the tissues and organs examined. Molecular phylogenetic analysis revealed that the cloned GnRHR belongs to the type 2/nonmammalian I GnRHR. Low-expression levels were observed from the pituitary glands of reproductively active leopard geckos, indicating the possibility that there is at least one more type of GnRHR highly expressed in the pituitary gland for the gonadotropin secretion in this reptile.

Structural determinants of agonist-induced signaling and regulation of the angiotensin AT 1 receptor

Angiotensin II (Ang II) regulates aldosterone secretion by stimulating inositol phosphate production and Ca 2+ signaling in adrenal glomerulosa cells via the G q-coupled AT 1 receptor, which is rapidly internalized upon agonist binding. Ang II also binds to the heptahelical AT 2 receptor, which neither activates inositol phosphate signaling nor undergoes receptor internalization. The differential behaviors of the AT 1 and AT 2 receptors were analyzed in chimeric angiotensin receptors created by swapping the second (IL2), the third (IL3) intracellular loops and/or the cytoplasmic tail (CT) between these receptors. When transiently expressed in COS-7 cells, the chimeric receptors showed only minor alterations in their ligand binding properties. Measurements of the internalization kinetics and inositol phosphate responses of chimeric AT 1A receptors indicated that the CT is required for normal receptor internalization, and IL2 is a determinant of G protein activation. In addition, the amino-terminal portion of IL3 is required for both receptor functions. However, only substitution of IL2 impaired Ang II-induced ERK activation, suggesting that alternative mechanisms are responsible for ERK activation in signaling-deficient mutant AT 1 receptors. Substitution of IL2, IL3, or CT of the AT 1A receptor into the AT 2 receptor sequence did not endow the latter with the ability to internalize or to mediate inositol phosphate signaling responses. These data suggest that the lack of receptor internalization and inositol phosphate signal generation by the AT 2 receptor is a consequence of its different activation mechanism, rather than the inability of its cytoplasmic domains to couple to intracellular effectors.

Characterization of angiotensin II–receptor subtypes in podocytes

Glomerular podocytes play a key role in maintaining the integrity of the glomerular filtration barrier. This function may be regulated by angiotensin II (Ang II) through activation of cell-surface receptors. Although studies suggest that podocytes express receptors for Ang II, the Ang II binding site has not been characterized with radioligand binding techniques. We therefore used iodine 125–labeled Ang II to monitor Ang II–receptor density during differentiation of a mouse podocyte cell line. Scatchard analyses of equilibrium binding data revealed a single class of high-affinity binding sites (dissociation constant ∼ 3 nmol/L) in both differentiated and nondifferentiated cells. During differentiation, the density of Ang II–receptor sites increased roughly 15-fold in differentiated podocytes (maximal density of specific binding sites 881 fmol/mg protein) compared with that in nondifferentiated cells (52 fmol/mg protein; P < .005). Glomerular podocytes expressed messenger RNA for AT1A, AT1B, and AT2 receptor subtypes, and competitive binding studies found that differentiated podocytes expressed mostly AT1 receptors (∼75%) with lesser amounts of AT2 (∼25%). Up-regulation of Ang II–receptor number was associated with increased Ang II–receptor responsiveness, as evidenced by enhanced Ang II–stimulated inositol phosphate (IP) generation and incorporation of tritiated thymidine. Both [ 3H]thymidine incorporation and IP generation were mediated by AT1-receptor activation. These data suggest that glomerular podocytes express a high-affinity binding site for Ang II with pharmacologic characteristics of both AT1 and AT2 receptors. This receptor site is up-regulated during podocyte differentiation, and receptor activation induces both IP generation and DNA synthesis by AT1-dependent mechanisms. We speculate that activation of podocyte Ang II receptors contributes to glomerular damage in disease states.

Signal transduction underlying carbachol-induced contraction of rat urinary bladder. I. Phospholipases and Ca2+ sources.

We have reexamined the muscarinic receptor subtype mediating carbachol-induced contraction of rat urinary bladder and investigated the role of phospholipase (PL)C, D, and A2 and of intra- and extracellular Ca2+ sources in this effect. Based on the nonsubtype-selective tolterodine, the highly M2 receptor-selective (R)-4-[2-[3-(4-methoxy-benzoylamino)-benzyl]-piperidin-1-ylmethyl]-piperidine-1-carboxylic acid amide (Ro-320-6206), and the highly M3 receptor-selective darifenacin and 3-(1-carbamoyl-1,1-diphenylmethyl)-1-(4-methoxyphenylethyl)pyrrolidine (APP), contraction occurs via M3 receptors. Carbachol stimulated inositol phosphate formation in rat bladder slices, and this was abolished by the phospholipase C inhibitor 1-(6-[([17beta]-3-methoxyestra-1,3,5[10]-trien-17-yl)-amino]hexyl)-1H-pyrrole-2,5-dione (U 73,122; 10 microM). Nevertheless, U 73,122 (1-10 microM) did not significantly affect carbachol-stimulated bladder contraction. Carbachol had only little effect on PLD activity in bladder slices, but the PLD inhibitor butan-1-ol, relative to its negative control butan-2-ol (0.3% each), caused detectable inhibition of carbachol-induced bladder contraction. The cytosolic PLA2 inhibitor arachidonyltrifluoromethyl ketone weakly inhibited carbachol-induced contraction at a concentration of 300 microM, but the cyclooxygenase inhibitor indomethacin (1-10 microM) remained without effect. The Ca2+ entry blocker nifedipine (10-100 nM) almost completely inhibited carbachol-induced bladder contraction. In contrast, 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole HCl (SKF 96,365; 10 microM), an inhibitor of store-operated Ca2+ channels, caused little inhibition. We conclude that carbachol-induced contraction of rat bladder largely depends on Ca2+ entry through nifedipine-sensitive channels and, perhaps, PLD, PLA2, and store-operated Ca2+ channels, whereas cyclooxygenase and, surprisingly, also PLC are not involved to a relevant extent.

Direct Activation of Phospholipase C-ϵ by Rho

Unique among the phospholipase C isozymes, the recently identified phospholipase C-epsilon (PLC-epsilon) contains an amino-terminal CDC25 domain capable of catalyzing nucleotide exchange on Ras family GTPases as well as a tandem array of Ras-associating (RA) domains near its carboxyl terminus that are effector binding sites for activated H-Ras and Rap. To determine whether other small GTPases activate PLC-epsilon, we measured inositol phosphate accumulation in COS-7 cells expressing a broad range of GTPase-deficient mutants of Ras superfamily proteins. RhoA, RhoB, and RhoC all markedly stimulated inositol phosphate accumulation in PLC-epsilon-expressing cells. This stimulation matched or exceeded phospholipase activation promoted by co-expression of PLC-epsilon with the known regulators Ras, Galpha12/13, or Gbeta1gamma2. In contrast, little effect was observed with the other Rho family members Rac1, Rac2, Rac3, and Cdc42. Truncation of the two carboxyl-terminal RA domains caused loss of responsiveness to H-Ras but not to Rho. Truncation of PLC-epsilon to remove the CDC25 and pleckstrin homology (PH) domains also did not cause loss of responsiveness to Rho, Galpha12/13, or Gbeta1gamma2. Comparative sequence analysis of mammalian phospholipase C isozymes revealed a unique approximately 65 amino acid insert within the catalytic core of PLC-epsilon not present in PLC-beta, gamma, delta, or zeta. A PLC-epsilon construct lacking this region was no longer activated by Rho or Galpha12/13 but retained regulation by Gbetagamma and H-Ras. GTP-dependent interaction of Rho with PLC-epsilon was illustrated in pull-down experiments with GST-Rho, and this interaction was retained in the PLC-epsilon construct lacking the unique insert within the catalytic core. These results are consistent with the conclusion that Rho family GTPases directly interact with PLC-epsilon by a mechanism independent of the CDC25 or RA domains. A unique insert within the catalytic core of PLC-epsilon imparts responsiveness to Rho, which may signal downstream of Galpha12/13 in the regulation of PLC-epsilon, because activation by both Rho and Galpha12/13 is lost in the absence of this sequence.

Proton-sensing G-protein-coupled receptors.

Blood pH is maintained in a narrow range around pH 7.4 mainly through regulation of respiration and renal acid extrusion. The molecular mechanisms involved in pH homeostasis are not completely understood. Here we show that ovarian cancer G-protein-coupled receptor 1 (OGR1), previously described as a receptor for sphingosylphosphorylcholine, acts as a proton-sensing receptor stimulating inositol phosphate formation. The receptor is inactive at pH 7.8, and fully activated at pH 6.8-site-directed mutagenesis shows that histidines at the extracellular surface are involved in pH sensing. We find that GPR4, a close relative of OGR1, also responds to pH changes, but elicits cyclic AMP formation. It is known that the skeleton participates in pH homeostasis as a buffering organ, and that osteoblasts respond to pH changes in the physiological range, but the pH-sensing mechanism operating in these cells was hitherto not known. We detect expression of OGR1 in osteosarcoma cells and primary human osteoblast precursors, and show that these cells exhibit strong pH-dependent inositol phosphate formation. Immunohistochemistry on rat tissue sections confirms the presence of OGR1 in osteoblasts and osteocytes. We propose that OGR1 and GPR4 are proton-sensing receptors involved in pH homeostasis.