Molecular cloning,functional expression,and gene silencing of two Drosophila receptors for the Drosophila neuropeptide pyrokinin-2

The database of the Drosophila Genome Project contains the sequences of two genes, CG8784 and CG8795, predicted to code for two structurally related G protein-coupled receptors. We have cloned these genes and expressed their coding parts in Chinese hamster ovary cells. We found that both receptors can be activated by low concentrations of the Drosophila neuropeptide pyrokinin-2 (CG8784, EC(50) for pyrokinin-2, 1x10(-9)M; CG8795, EC(50) for pyrokinin-2, 5 x 10(-10)M). The precise role of Drosophila pyrokinin-2 (SVPFKPRLamide) in Drosophila is unknown, but in other insects, pyrokinins have diverse myotropic actions and are also initiating sex pheromone biosynthesis and embryonic diapause. Gene silencing, using the RNA-mediated interference technique, showed that CG8784 gene silencing caused lethality in embryos, whereas CG8795 gene silencing resulted in strongly reduced viability for both embryos and first instar larvae. In addition to the two Drosophila receptors, we also identified two probable pyrokinin receptors in the genomic database from the malaria mosquito Anopheles gambiae. The two Drosophila pyrokinin receptors are, to our knowledge, the first invertebrate pyrokinin receptors to be identified.     

Molecular identification of a Drosophila G protein-coupled receptor specific for crustacean cardioactive peptide

The Drosophila Genome Project website (www.flybase.org) contains the sequence of an annotated gene (CG6111) expected to code for a G protein-coupled receptor. We have cloned this receptor and found that its gene was not correctly predicted, because an annotated neighbouring gene (CG14547) was also part of the receptor gene. DNA corresponding to the corrected gene CG6111 was expressed in Chinese hamster ovary cells, where it was found to code for a receptor that could be activated by low concentrations of crustacean cardioactive peptide, which is a neuropeptide also known to occur in Drosophila and other insects (EC(50), 5.4 x 10(-10)M). Other known Drosophila neuropeptides, such as adipokinetic hormone, did not activate the receptor. The receptor is expressed in all developmental stages from Drosophila, but only very weakly in larvae. In adult flies, the receptor is mainly expressed in the head. Furthermore, we identified a gene sequence in the genomic database from the malaria mosquito Anopheles gambiae that very likely codes for a crustacean cardioactive peptide receptor.     

Molecular identification of the first SIFamide receptor

SIFamide is the short name and also the C terminus of the Drosophila neuropeptide AYRKPPFNGSIFamide. SIFamide has been isolated or predicted from various insects and crustaceans, and appears to be extremely well conserved among these arthropods. However, the function of this neuropeptide is still enigmatic. Here, we have identified the Drosophila gene (CG10823) coding for the SIFamide receptor. When expressed in Chinese hamster ovary cells, the receptor is only activated by Drosophila SIFamide (EC(50), 2x10(-8)M) and not by a library of 32 other insect neuropeptides and eight biogenic amines. Database searches revealed SIFamide receptor orthologues in the genomes from the malaria mosquito Anopheles gambiae, the silkworm Bombyx mori, the red flour beetle Tribolium castaneum, and the honey bee Apis mellifera. An alignment of the five insect SIFamide or SIFamide-like receptors showed, again, an impressive sequence conservation (67-77% amino acid sequence identities between the seven-transmembrane areas; 82-87% sequence similarities). The identification of well-conserved SIFamide receptor orthologues in all other insects with a sequenced genome, suggests that the SIFamide/receptor couple must have an essential function in arthropods. This paper is the first report on the identification of a SIFamide receptor.     

Molecular identification of a myosuppressin receptor from the malaria mosquito Anopheles gambiae

The insect myosuppressins (X1DVX2HX3FLRFamide) are neuropeptides that generally block insect muscle activities. We have used the genomic sequence information from the malaria mosquito Anopheles gambiae Genome Project to clone a G protein-coupled receptor that was closely related to the two previously cloned and characterized myosuppressin receptors from Drosophila [Proc. Natl. Acad. Sci. USA 100 (2003) 9808]. The mosquito receptor cDNA was expressed in Chinese hamster ovary cells and was found to be activated by low concentrations of Anopheles myosuppressin (TDVDHVFLRFamide; EC50, 1.6 x 10(-8)M). The receptor was not activated by a library of 35 other insect neuropeptides and monoamines, including neuropeptides that resembled myosuppressin in their C-terminal moiety, such as PDRNFLRFamide (Anopheles FMRFamide-3), other Anopheles FMRFamide peptides, or neuropeptide F-like peptides, showing that the receptor was quite selective for myosuppressin. These results also showed that the myosuppressin receptor needs a much larger portion than the C-terminal FLRFamide sequence for its activation. The insect myosuppressins are often grouped together with the insect FMRFamides under the name FaRPs (FMRFamide-related peptides). However, this is not justified anymore, because the insect myosuppressin receptor/ligand couple is both functionally and evolutionarily fully unrelated to the insect FMRFamide receptor/ligand couple. To our knowledge, this is the first report on the molecular identification of a mosquito neuropeptide receptor.     

A new family of insect tyramine receptors

The Drosophila Genome Project database contains a gene, CG7431, annotated to be an "unclassifiable biogenic amine receptor." We have cloned this gene and expressed it in Chinese hamster ovary cells. After testing various ligands for G protein-coupled receptors, we found that the receptor was specifically activated by tyramine (EC(50), 5x10(-7)M) and that it showed no cross-reactivity with beta-phenylethylamine, octopamine, dopa, dopamine, adrenaline, noradrenaline, tryptamine, serotonin, histamine, and a library of 20 Drosophila neuropeptides (all tested in concentrations up to 10(-5) or 10(-4)M). The receptor was also expressed in Xenopus oocytes, where it was, again, specifically activated by tyramine with an EC(50) of 3x10(-7)M. Northern blots showed that the receptor is already expressed in 8-hour-old embryos and that it continues to be expressed in all subsequent developmental stages. Adult flies express the receptor both in the head and body (thorax/abdomen) parts. In addition to the Drosophila tyramine receptor gene, CG7431, we found another closely related Drosophila gene, CG16766, that probably also codes for a tyramine receptor. Furthermore, we annotated similar tyramine-like receptor genes in the genomic databases from the malaria mosquito Anopheles gambiae and the honeybee Apis mellifera. These four tyramine or tyramine-like receptors constitute a new receptor family that is phylogenetically distinct from the previously identified insect octopamine/tyramine receptors. The Drosophila tyramine receptor is, to our knowledge, the first cloned insect G protein-coupled receptor that appears to be fully specific for tyramine.     

Molecular cloning and functional expression of a Drosophila receptor for the neuropeptides capa-1 and -2

The Drosophila Genome Project website contains an annotated gene (CG14575) for a G protein-coupled receptor. We cloned this receptor and found that the cloned cDNA did not correspond to the annotated gene; it partly contained different exons and additional exons located at the 5(')-end of the annotated gene. We expressed the coding part of the cloned cDNA in Chinese hamster ovary cells and found that the receptor was activated by two neuropeptides, capa-1 and -2, encoded by the Drosophila capability gene. Database searches led to the identification of a similar receptor in the genome from the malaria mosquito Anopheles gambiae (58% amino acid residue identities; 76% conserved residues; and 5 introns at identical positions within the two insect genes). Because capa-1 and -2 and related insect neuropeptides stimulate fluid secretion in insect Malpighian (renal) tubules, the identification of this first insect capa receptor will advance our knowledge on insect renal function.     

Cloning and characterization of the adipokinetic hormone receptor from the cockroach Periplaneta americana

Cockroaches have long been used as insect models to investigate the actions of biologically active neuropeptides. Here, we describe the cloning and functional expression in Chinese hamster ovary cells of an adipokinetic hormone (AKH) G protein-coupled receptor from the cockroach Periplaneta americana. This receptor is only activated by various insect AKHs (we tested eight) and not by a library of 29 other insect or invertebrate neuropeptides and nine biogenic amines. Periplaneta has two intrinsic AKHs, Pea-AKH-1, and Pea-AKH-2. The Periplaneta AKH receptor is activated by low concentrations of both Pea-AKH-1 (EC50, 5 x 10(-9)M), and Pea-AKH-2 (EC50, 2 x 10(-9)M). Insects can be subdivided into two evolutionary lineages, holometabola (insects with a complete metamorphosis during development) and hemimetabola (incomplete metamorphosis). This paper describes the first AKH receptor from a hemimetabolous insect.     

Aryl hydrocarbon receptor response to indigoids in vitro and in vivo

Indigo and indirubin have been reported to be present at low levels in human urine. The possibility that indigoids are physiological ligands of the aryl hydrocarbon receptor (AhR) has been suggested by initial studies in yeast, where indirubin was found to be 50 times more potent than 2,3,7,8-tetrachlorodibenzo[p]dioxin (TCDD), and indigo was found to be equipotent. To demonstrate that these indigoids are bona fide agonists in mammalian systems, we employed a number of in vitro and in vivo measures of AhR agonist potency. In a hepatoma cell reporter system, indigo yielded an EC50 of approximately 5x10(-6)M (indirubin 3' -oxime EC50 approximately 5x10(-7)M, indirubin EC50 approximately 1x10(-7)M). A comparison of these EC50 values with that of 2,3,7,8-tetrachlorodibenzofuran (TCDBF) ( approximately 3x10(-9)M) indicated that these compounds are less potent than classic halogenated-dibenzofurans or -dibenzo-p-dioxins. Competitive binding assays for AhR occupancy showed similar IC50 values for indirubin and TCDBF ( approximately 2x10(-9) and 5x10(-9)M), with the IC50 values of indigo and indirubin 3' -oxime being approximately 10-fold higher. When rats were treated with these indigoids in the range of 1.5-50mg/kg, induction of hepatic cytochrome P450 1A1 was detected. Differences in the rank-order of potency observed in vivo and in vitro could, in part, be explained by metabolism. Although their biological potencies are not as high as has been previously suggested, collectively the results show that these indole-derived pigments are agonists of AhR in vivo. The in vivo results suggest that solubility, distribution, and metabolism influence the response to the compounds.     

Adenosine A 2B receptors modulate cAMP levels and induce CREB but not ERK1/2 and p38 phosphorylation in rat skeletal muscle cells

The present study examined the existence of the adenosine A(1),A(2A), and A(2B) receptors and the effect of receptor activation on cAMP accumulation and protein phosphorylation in primary rat skeletal muscle cells. Presence of mRNA and protein for all three receptors was demonstrated in both cultured and adult rat skeletal muscle. NECA (10(-9)-10(-4)M) increased the cAMP concentration in cultured muscle cells with an EC(50) of (95% confidence interval)=15 (5.9-25.1) micro M, whereas CGS 21680 (10(-9)-10(-4)M) had no effect on cAMP accumulation. Concentrations of [R]-PIA below 10(-6)M had no effect on cAMP accumulation induced by either isoproterenol or forskolin. NECA resulted in phosphorylation of CREB with an EC(50) of (95% confidence interval)=1.7 (0.40-7.02) micro M, whereas ERK1/2 and p38 phosphorylation was unchanged. The results show that, although the A(1),A(2A), and A(2B) receptors are all present in skeletal muscle cells, the effect of adenosine on adenylyl cyclase activation and phosphorylation of CREB is mainly mediated via the adenosine A(2B) receptor.     

Isolation and characterization of antagonist and agonist peptides to the human melanocortin 1 receptor

We identified a large number of peptide mimotopes of the adrenocorticotropic hormone (ACTH) and the alpha-melanocyte stimulating hormone (alpha-MSH) to analyze better the structure-function relationships of these hormones with the human MC1 receptor (hMC1R). We have investigated the use of phage-display technology to isolate specific peptides of this receptor by using three monoclonal anti-ACTH antibodies (mAbs). A library of 10(8) phage-peptides displaying randomized decapeptides was constructed and used to select phage-peptides that bind to mAbs. Forty-five phage-peptides have been isolated and from their amino acid sequences, we have identified two consensus sequences, EXFRWGKPA and WGXPVGKP, corresponding to the regions 5-13 and 9-16 of ACTH, respectively. A biological assay on cells expressing the hMC1-R was developed to determine the capacity of phage-peptides to stimulate the receptor. Only two phage-peptides showed detectable activity. Thirty-one peptides were synthesized to analyze their biological effect. We identified two weak agonists, EC50=16 and 11 microM, two strong agonists, EC50=25 and 14 nM and a partial antagonist, IC50=36 microM. This work confirmed the modulator agonist role of the regions 11-12 of alpha-MSH and ACTH, and the importance of the methionine residue at position 4 for the stimulation of the hMC1-R. We also identified analogues of the regions 8-17 of ACTH that exhibited a weak activator effect, and of one analogue of the N-terminal regions 1-9 of ACTH and alpha-MSH having a partial antagonist effect. These results may be useful in the development of potential agonists or antagonists of the hMC1R.     

Cytosolic Ca2+ responses to sub-picomolar and nanomolar PACAP in pancreatic beta-cells are mediated by VPAC2 and PAC1 receptors

Pituitary adenylate cyclase-activating polypeptide (PACAP) potentiates glucose-induced insulin release and increases cytosolic Ca2+ concentration ([Ca2+]i) in islet beta-cells in a concentration-dependent manner with two peaks at 10(-13) and 10(-9) M. PAC1 receptor (PAC1-R) and VPAC2 receptor (VPAC2-R) are expressed in pancreatic beta-cells and thought to be involved in insulin release. We aimed to determine the receptor types involved in the [Ca2+]i responses to 10(-13) and 10(-9) M PACAP. We measured [Ca2+]i in beta-cells and examined comparative effects of PAC1-R-selective agonist maxadilan, its antagonist M65, VPAC2-R-selective agonist Ro25-1553, and native ligands of PACAP and VIP. In the presence of 8.3 mM glucose, maxadilan, Ro25-1553, PACAP, and VIP at 10(-13) and 10(-9) M all increased [Ca2+]i. PACAP and maxadilan elicited greater effects at 10(-9) M than at 10(-13) M both in the incidence and amplitude of [Ca2+]i responses. For VIP and Ro25-1553, in contrast, the effects at 10(-9) and 10(-13) M were comparable. Furthermore, the amplitude of [Ca2+]i responses to 10(-9) M PACAP, but not 10(-13) M PACAP, was suppressed by M65. The results suggest that VPAC2-R and PAC1-R contribute equally to [Ca2+]i responses to sub-picomolar concentrations of PACAP, while PAC1-R has greater contribution to [Ca2+]i responses to nanomolar concentrations of this peptide.     

The Drosophila genes CG14593 and CG30106 code for G-protein-coupled receptors specifically activated by the neuropeptides CCHamide-1 and CCHamide-2

Recently, a novel neuropeptide, CCHamide, was discovered in the silkworm Bombyx mori (L. Roller et al., Insect Biochem. Mol. Biol. 38 (2008) 1147–1157). We have now found that all insects with a sequenced genome have two genes, each coding for a different CCHamide, CCHamide-1 and -2. We have also cloned and deorphanized two Drosophila G-protein-coupled receptors (GPCRs) coded for by genes CG14593 and CG30106 that are selectively activated by Drosophila CCH-amide-1 (EC₅₀, 2 × 10⁻⁹ M) and CCH-amide-2 (EC₅₀, 5 × 10⁻⁹ M), respectively. Gene CG30106 (symbol synonym CG14484) has in a previous publication (E.C. Johnson et al., J. Biol. Chem. 278 (2003) 52172–52178) been wrongly assigned to code for an allatostatin-B receptor. This conclusion is based on our findings that the allatostatins-B do not activate the CG30106 receptor and on the recent findings from other research groups that the allatostatins-B activate an unrelated GPCR coded for by gene CG16752. Comparative genomics suggests that a duplication of the CCHamide neuropeptide signalling system occurred after the split of crustaceans and insects, about 410 million years ago, because only one CCHamide neuropeptide gene is found in the water flea Daphnia pulex (Crustacea) and the tick Ixodes scapularis (Chelicerata).     

Characterization of functional histamine H1 receptors on a cultured smooth muscle cell line

Cultured cells of the smooth muscle line DDT1MF-2, which was derived from a hamster vas deferens tumor, expressed histamine H1-type receptors and responded biochemically and functionally to H1-specific stimulation. The H1-receptor antagonist [3H]-pyrilamine bound specifically to 9.7 x 10(6) sites/DDT1MF-2 cell with a dissociation constant (Kd) of 219 nM. The addition of histamine to suspensions of fura-2-loaded DDT1MF-2 cells elicited a rapid, transient, and stimulus concentration-dependent increase in the intracellular concentration of Ca2+ with an EC50 of 3 x 10(-5) M, which demonstrated H1 receptor specificity. Moreover, in order to evaluate in vitro contractile response of individual DDT1MF-2 cells, the degree of intracellular actin polymerization was quantified by a DNase inhibition assay. The percentage of nonpolymerized or G-actin in DDT1MF-2 cells was reduced in a histamine concentration-dependent manner with an EC50 of 1 x 10(-5) M and H1 receptor specificity. Histamine-induced actin polymerization was accompanied by changes in cell shape that were consistent with cellular contraction, as assessed by flow cytometry. The H1-type receptors of cultured DDT1MF-2 cells thus couple histamine stimulation to a variety of functional responses of smooth muscle cells.     

Identification of four evolutionarily related G protein-coupled receptors from the malaria mosquito Anopheles gambiae

The mosquito Anopheles gambiae is an important vector for malaria, which is one of the most serious human parasitic diseases in the world, causing up to 2.7 million deaths yearly. To contribute to our understanding of A. gambiae and to the transmission of malaria, we have now cloned four evolutionarily related G protein-coupled receptors (GPCRs) from this mosquito and expressed them in Chinese hamster ovary cells. After screening of a library of thirty-three insect or other invertebrate neuropeptides and eight biogenic amines, we could identify (de-orphanize) three of these GPCRs as: an adipokinetic hormone (AKH) receptor (EC₅₀ for A. gambiae AKH, 3 × 10⁻⁹ M), a corazonin receptor (EC₅₀ for A. gambiae corazonin, 4 × 10⁻⁹ M), and a crustacean cardioactive peptide (CCAP) receptor (EC₅₀ for A. gambiae CCAP, 1 × 10⁻⁹ M). The fourth GPCR remained an orphan, although its close evolutionary relationship to the A. gambiae and other insect AKH receptors suggested that it is a receptor for an AKH-like peptide. This is the first published report on evolutionarily related AKH, corazonin, and CCAP receptors in mosquitoes.     

Purification, structural characterization, and myotropic activity of a peptide related to des-Arg(9)-bradykinin from an elasmobranch fish, the little skate, Leucoraja erinacea

A bradykinin (BK)-related peptide was isolated from heat-denaturated plasma from an elasmobranch fish, the little skate, Leucoraja erinacea after incubation with porcine pancreatic kallikrein. The primary structure of the peptide (H-Gly-Ile-Thr-Ser-Trp-Leu-Pro-Phe-OH; skate BK) shows limited structural similarity to the mammalian B1 receptor agonist, des-Arg(9)-BK. The myotropic activities of synthetic skate BK, and the analog skate [Arg(9)]BK, were examined in isolated skate vascular and intestinal smooth muscle preparations. Skate BK produced a concentration-dependent constriction of the mesenteric artery (EC(50)=4.37x10(-8)M; maximum response=103.4+/-10.23% of the response to 60mM KCl) but the response to skate [Arg(9)]BK was appreciably weaker (response to 10(-6)M=73.0+/-23.4% of the response to 60mM KCl). Neither the first branchial gill arch nor the ventral aorta responded to either purified peptide. Skate BK also produced a concentration-dependent constriction of intestinal smooth muscle preparations (EC(50)=2.74x10(-7)M; maximum response 31.0+/-12.2% of the response to 10(-5)M acetylcholine). Skate [Arg(9)]BK was without effect on the intestinal preparation. The data provide evidence for the existence of the kallikrein-kinin system in a phylogenetically ancient vertebrate group and the greater potency of skate BK compared with the analog skate [Arg(9)]BK suggests that the receptor mediating vascular responses resembles the mammalian B1 receptor more closely than the B2 receptor.     

ETB receptor dependent alteration in aortic responses to ET-1 in the cardiomyopathic hamster

The aim of this study was to verify whether an alteration in the aortic endothelin-1 (ET-1) response takes place in UM-X7.1 cardiomyopathic hamsters. Our results showed that ET-1 (10(-12) - 10(-5) mol/L) induces dose-dependent sustained increases in tension in the intact and endothelium denuded aortas from both normal and cardiomyopathic hamsters. The EC50 values of ET-1 of both intact and endothelium denuded aortas of normal hamsters were similar (2.2 x 10(-9) mol/L and 1.8 x 10(-9) mol/L, respectively). However, in cardiomyopathic hamsters, the EC50 of ET-1 in intact aortas was higher (1.5 x 10(-8) mol/L) than that of the endothelium denuded preparations (2.7 x 10(-9) mol/L). The EC50 of ET-1 in normal and cardiomyopathic hamster denuded aortas were similar. However, the EC50 of ET-1 in intact aortas of cardiomyopathic hamster was higher (1.5 x 10(-8) mol/L) than that of normal hamsters (2.2 x 10(-9) mol/L). Pre-treatment with the ETA receptor antagonist ABT-627 (10(-5)mol/L) of intact and endothelium denuded aortas from both normal and cardiomyopathic hamsters significantly prevented ET-1 (10(-7) mol/L) from inducing an increase in tension. Pre-treatment with the ETB receptor antagonist A-192621 (10(-5) mol/L) had no effect on the ET-1-induced increase in tension in endothelium denuded aortas of both normal and cardiomyopathic hamsters, as well as in intact preparations of normal animals. However, blockade of the ETB receptors in intact aortas of cardiomyopathic hamsters significantly (p < 0.001) potentiated the ET-1-induced increase in tension. In summary, an attenuation of the contraction response to ET-1 was found in UM-X7.1 cardiomyopathic hamsters when compared with normal age-matched hamsters. This alteration of the ET-1 effect in the aortas of cardiomyopathic hamsters seems to be dependent on the presence of the endothelium and could be due, in part, to an increase in the contribution of endothelial ETB receptors to relaxation, which in turn acts as a physiological depressor of ET-1 vasoconstriction. Our results suggest that an increase in the endothelium ETB receptor density may play a role in the development of hypotension in UM-X7.1 cardiomyopathic hamsters.     

Effects of the thromboxane receptor antagonist SK&F 88046 in the canine, monkey and human coronary vasculature

U-46619, a stable "functional" thromboxane/endoperoxide receptor agonist, produced potent contractile responses in isolated canine, rhesus monkey and human left circumflex coronary arteries (EC50 = 9.11 x 10(-9)M, 1.98 x 10(-8)M and 3.50 x 10(-9)M, respectively). Canine intrapulmonary veins were also contracted potently by U-46619 (EC50 = 1.22 x 10(-9)M). SK&F 88046, a thromboxane A2 (TxA2) end-organ receptor antagonist, blocked the vasoconstrictor effects of U-46619 in the canine circumflex artery (KB = 1.33 x 10(-8)M), canine intrapulmonary vein (KB = 1.46 x 10(-9)M), monkey circumflex artery (KB = 8.47 x 10(-8)M), and human circumflex artery (KB = 8.49 x 10(-7)M). SK&F 88046 was 10-60 times more potent in the canine and rhesus monkey coronary vasculature than in the human coronary preparations. Intracoronary administration of U-46619 to anesthetized, open chest dogs produced a dose-related decrease in left circumflex coronary artery blood flow which resulted in decreases in left ventricular developed pressure, left ventricular positive and negative dP/dt, ascending aortic blood flow, and an increase in left ventricular end-diastolic pressure. The decrease in coronary blood flow and the hemodynamic changes were either attenuated or completely inhibited by i.v. administration of SK&F 88046 (2.5 mg/kg + 0.05 mg/kg/min or 5.0 mg/kg + 0.1 mg/kg/min). SK&F 88046 was compared to two other TxA2 receptor antagonists in canine isolated intrapulmonary veins. SQ 29,548 was approximately 2-times more potent than SK&F 88046 as an antagonist of U-44619 mediated contractions (KB = 7.0 x 10(-10)M). In contrast, BM 13.177 was 150-fold less potent (KB = 2.19 x 10(-7)M) than SK&F 88046. Thus, the present study demonstrates species variability in response to TxA2 agonists and antagonists and reconfirms the relative importance of species selection in studying these agents.     

Human osteoblast-like cell proliferation induced by calcitonin-related peptides involves PKC activity

The calcitonin peptides [calcitonin (CT), calcitonin gene-related peptide (CGRP), amylin] share many biological actions, including activity on bone cells. In the present study, CT (10(-11) to 10(-9) M) stimulated [(3)H]thymidine incorporation in primary cultures of human osteoblasts (hOB), as already demonstrated for CGRP and amylin. RT-PCR analysis showed that the calcitonin receptor and the calcitonin receptor-like receptor are both expressed in hOB. In these cells, CT (10(-10) M) and amylin (10(-9) M), in contrast to CGRP (10(-8) M), did not increase cAMP production. All three peptides stimulated protein kinase C (PKC) activity. To evaluate PKC involvement in hOB proliferation, cells were incubated with phorbol 12,13-dibutyrate, a stimulator of PKC activity; cell proliferation was increased in a dose-dependent manner (EC(50) = 3.4 x 10(-8) M). Staurosporine (10(-9) M), a PKC inhibitor, blocked phorbol 12,13-dibutyrate-induced PKC activity and cell proliferation. Inhibition of PKC by staurosporine also counteracted the stimulatory effect of CT, CGRP, and amylin on hOB proliferation. From these data, it is deduced that the activation of PKC is important for hOB proliferation and that it is involved in the anabolic effect of CT peptides on bone.     

Prostaglandin E2 receptor heterogeneity and dysfunction in mammary tumor cells

We have reported previously that murine mammary tumor cell subpopulations isolated from one spontaneous adenocarcinoma are heterogenous in terms of prostaglandin E2 (PGE2) synthetic capacity. We have also shown that tumor-PGE2 contributes to the ability of these cells to grow and metastasize in vivo (Fulton and Heppner: Cancer Research 45:4779-4784, 1985). In the present study, we have asked whether exogenous PGE2 has direct effects on the proliferation of these cells in vitro and if such responses can be attributed to the capacity of these cells to 1) bind PGE2 and 2) activate adenylate cyclase via the PGE2 receptor. We report that PGE2, at concentrations below 1 x 10(-5) M, does not affect the proliferation rate of these cells. This unresponsiveness is not due to the absence of receptors for PGE2. However, marked heterogeneity in receptor binding and function was detected in these closely related cell lines. Two metastatic lines (66 and 410.4) have high-affinity receptors for PGE2 (average Kd = 4.3 x 10(-9) M/L and 4.2 x 10(-9) M/L, respectively) and similar binding capacities (4.1 x 10(-4) and 2.9 x 10(4) binding sites, respectively). Two nonmetastatic lines, 410 and 67, have receptors with lower affinity (Kd = 8.3 x 10(-9) M/L and 1.6 x 10(-7) M/L, respectively) and binding capacities of 2.8 x 10(5)/410 cell or 7.3 x 10(4)/67 cell. A third nonmetastatic line (168) exhibits no specific binding. PGE2 receptor stimulation leads to elevated intracellular cAMP in lines 66, 410, and 67. Line 410.4 cells appear to have a functional lesion in the PGE2 receptor resulting in a failure to elevate cAMP in response to receptor occupancy. Adenylate cyclase can, however, be activated in these cells by cholera toxin, NaF, or forskolin. In comparison to the other cell lines, line 168 cells respond poorly to all cAMP-stimulating agents. Thus, we have found that PGE2 binding is a heterogenous property for these cells, and, in addition, we have identified an apparent uncoupling of PGE2 receptor to the adenylate cyclase system in one cell line.