Stimulation Of Cyclic AMP Production Research Articles

Structure-based redesign of an edema toxin inhibitor

Edema factor (EF) toxin of Bacillus anthracis (NIAID category A), and several other toxins from NIAID category B Biodefense target bacteria are adenylyl cyclases or adenylyl cyclase agonists that catalyze the conversion of ATP to 3′,5′-cyclic adenosine monophosphate (cAMP). We previously identified compound 1 (3-[(9-oxo-9H-fluorene-1-carbonyl)-amino]-benzoic acid), that inhibits EF activity in cultured mammalian cells, and reduces diarrhea caused by enterotoxigenic Escherichia coli (ETEC) at an oral dosage of 15μg/mouse. Here, molecular docking was used to predict improvements in potency and solubility of new derivatives of compound 1 in inhibiting edema toxin (ET)-catalyzed stimulation of cyclic AMP production in murine monocyte-macrophage cells (RAW 264.7). Structure–activity relationship (SAR) analysis of the bioassay results for 22 compounds indicated positions important for activity. Several derivatives demonstrated superior pharmacological properties compared to our initial lead compound, and are promising candidates to treat anthrax infections and diarrheal diseases induced by toxin-producing bacteria.

ORIGINAL ARTICLE: Monoclonal autoantibodies to the TSH receptor, one with stimulating activity and one with blocking activity, obtained from the same blood sample

SummaryObjective Patients who appear to have both stimulating and blocking TSHR autoantibodies in their sera have been described, but the two activities have not been separated and analysed. We now describe the isolation and detailed characterization of a blocking type TSHR monoclonal autoantibody and a stimulating type TSHR monoclonal autoantibody from a single sample of peripheral blood lymphocytes.Design, patients and measurements Two heterohybridoma cell lines secreting TSHR autoantibodies were isolated using standard techniques from the lymphocytes of a patient with hypothyroidism and high levels of TSHR autoantibodies (160 units/l by inhibition of TSH binding). The ability of the two new monoclonal antibodies (MAbs; K1‐18 and K1‐70) to bind to the TSHR and compete with TSH or TSHR antibody binding was analysed. Furthermore, the effects of K1‐18 and K1‐70 on cyclic AMP production in Chinese hamster ovary cells (CHO) cells expressing the TSHR were investigated.Results One MAb (K1‐18) was a strong stimulator of cyclic AMP production in TSHR‐transfected CHO cells and the other (K1‐70) blocked stimulation of the TSHR by TSH, K1‐18, other thyroid‐stimulating MAbs and patient serum stimulating type TSHR autoantibodies. Both K1‐18 (IgG1 kappa) and K1‐70 (IgG1 lambda) bound to the TSHR with high affinity (0·7 × 1010 l/mol and 4 × 1010 l/mol, respectively), and this binding was inhibited by unlabelled K1‐18 and K1‐70, other thyroid‐stimulating MAbs and patient serum TSHR autoantibodies with stimulating or blocking activities. V region gene analysis indicated that K1‐18 and K1‐70 heavy chains used the same V region germline gene but different D and J germline genes as well as having different light chains. Consequently, the two antibodies have evolved separately from different B cell clones.Conclusions This study provides proof that a patient can produce a mixture of blocking and stimulating TSHR autoantibodies at the same time.

A Human Monoclonal Autoantibody to the Thyrotropin Receptor with Thyroid-Stimulating Blocking Activity

Human monoclonal autoantibodies (MAbs) are valuable tools to study autoimmune responses. To date only one human MAb to the thyrotropin (TSH) receptor (TSHR) with stimulating activity has been available. We now describe the detailed characterization of a blocking type human MAb to the TSHR. A single heterohybridoma cell line was isolated from the peripheral blood lymphocytes of a patient with severe hypothyroidism (TSH 278 mU/L) using standard techniques. The line stably expresses a TSHR autoantibody (5C9; IgG1/kappa). Ability of 5C9 to bind and compete with 125I-TSH or TSHR antibodies binding to the TSHR was tested using tubes coated with solubilized TSHR. Furthermore, the blocking effects of 5C9 on stimulation of cyclic AMP production was assessed using Chinese hamster ovary (CHO) cells expressing the wild-type human TSHR or TSHRs with amino acid mutations. 5C9 IgG bound to the TSHR with high affinity (4 x 10(10) L/mol) and inhibited binding of TSH and a thyroid-stimulating human monoclonal autoantibody (M22) to the receptor. 5C9 IgG preparations inhibited the cyclic AMP-stimulating activities of TSH, M22, serum TSHR autoantibodies and thyroid-stimulating mouse monoclonal antibodies. Furthermore 5C9 reduced the constitutive activity of wild-type TSHR and TSHR with some activating mutations. The effect of different amino acid mutations in the TSHR on 5C9 biological activity was studied and TSHR Lys129Ala or Asp203Ala completely abolished the ability of 5C9 to block TSH-mediated stimulation of cyclic AMP production. The availability of 5C9 provides new opportunities to investigate the binding and biological activity of TSHR blocking type autoantibodies including studies at the molecular level. Furthermore, monoclonal antibodies such as 5C9 may well provide the basis of new drugs to control TSHR activity including applications in thyroid cancer and Graves' ophthalmopathy.

Stimulation of cyclic AMP production by the Caenorhabditis elegans muscarinic acetylcholine receptor GAR-3 in Chinese hamster ovary cells

Among the three G-protein-linked acetylcholine receptors (GARs) in Caenorhabditis elegans ( C. elegans), GAR-3 is structurally and pharmacologically most similar to mammalian muscarinic acetylcholine receptors (mAChRs). Using Chinese hamster ovary (CHO) cells stably expressing GAR-3b, the major alternatively spliced isoform of GAR-3, we observed that carbachol stimulated cyclic AMP (cAMP) production in a dose- and time-dependent manner. The stimulating effect of carbachol was abolished by atropine, a muscarinic antagonist, indicating that the cAMP production is specifically mediated by GAR-3b. When the cells were treated with BAPTA-AM and EGTA, which reduce the cytosolic Ca 2+ level, carbachol-stimulated cAMP accumulation was inhibited by approximately 56%. Inhibition of protein kinase C (PKC) by chronic treatment with phorbol 12-myristate 13-acetate (PMA) or by GF109203X decreased carbachol-stimulated cAMP production by as much as 68%. It thus appears that Ca 2+ and PKC are critically involved in GAR-3b-mediated cAMP formation. We also observed that carbachol-stimulated cAMP production was further enhanced by pertussis toxin (PTX) treatment. This observation indicates that GAR-3b couples to a PTX-sensitive G protein, presumably Gi, to attenuate the cAMP accumulation. Taken together, our data show that GAR-3b stimulates cAMP production in CHO cells and suggest that GAR-3b couples to both stimulatory and inhibitory pathways to modulate the intracellular cAMP level.

Rapid stimulation of cyclic AMP production by aldosterone in rat inner medullary collecting ducts.

Aldosterone stimulates sodium transport in the inner medullary collecting duct (IMCD) via the classic genomic pathway, but it is not known whether it also acts via a rapid, non-conventional pathway in this part of the nephron. The IMCD regulates the final sodium content of urine and expresses vasopressin receptors coupled to adenylate cyclase. The recently reported rapid, non-genomic actions of aldosterone have been associated mainly with an increase in intracellular Ca(2+); however, it has also been shown to stimulate camp generation. Thus the aim of this study was to determine whether aldosterone stimulates rapid generation of cAMP in isolated IMCD segments. IMCD segments were microdissected from Sprague-Dawley rat kidneys and incubated at 37 degrees C for 4 min with aldosterone (10(-12) to 10(-6) M), vasopressin (10(-12) to 10(-6) M), or a combination of hormones in the presence of a phosphodiesterase inhibitor. cAMP was measured by radioimmunoassay. While corticosterone and dexamethasone were ineffective, aldosterone stimulated a dose-dependent increase in cAMP within 4 min (P<0.05). This action of aldosterone was not inhibited by the MR antagonist spironolactone. Co-incubation of aldosterone with vasopressin resulted in a further increase in cAMP generation above that induced by the neurohypophysial hormone alone. Aldosterone-mediated cAMP generation was not inhibited by a vasopressin V(1) or V(2) receptor antagonist. These data support a novel and rapid, non-genomic effect of aldosterone in IMCD. Aldosterone does not apparently interact with the vasopressin receptor to stimulate cAMP generation.

2-Alkynyl-8-aryl-9-methyladenines as novel adenosine receptor antagonists: their synthesis and structure-activity relationships toward hepatic glucose production induced via agonism of the A(2B) receptor.

Novel adenosine antagonists, 2-alkynyl-8-aryl-9-methyladenine derivatives, were synthesized as candidate hypoglycemic agents. These analogues were evaluated for inhibitory activity on N-ethylcarboxamidoadenosine (NECA)-induced glucose production in primary cultured rat hepatocytes. In general, aromatic moieties at the 8-position and alkynyl groups at the 2-position had significantly increased activity compared to unsubstituted compounds. The preferred substituents at the 8-position of adenine were the 2-furyl and 3-fluorophenyl groups. In modifying the alkynyl side chain, change of the ring size, cleavage of the ring, and removal of the hydroxyl group were well tolerated. The order of the stimulatory effects of adenosine agonists on rat hepatocytes was NECA > CPA > CGS21680, which is consistent with involvement of the A(2B) receptor. In Chinese hamster ovary cells stably transfected with human A(2B) receptor cDNA, one of the compounds potent in hepatocytes, 15o (IC(50) = 0.42 microM), antagonized NECA-induced stimulation of cyclic AMP production (IC(50) = 0.063 microM). This inhibitory effect was much more potent than those of FK453, KF17837, and L249313 which have been reported to be respectively A(1), A(2A), and A(3) selective antagonists. These findings agree very well with the result that, compared to 15o, these selective antagonists for each receptor subtype showed only marginal effects in rat hepatocytes. These results suggest that adenosine agonist-induced glucose production in rat hepatocytes is mediated through the A(2B) receptor. Furthermore, 15o showed hypoglycemic activity in an animal model of noninsulin-dependent diabetes mellitus, the KK-A(y) mice. It is possible that inhibition of hepatic glucose production via the A(2B) receptor could be at least one of the mechanisms by which 15o exerts its in vivo effects. Further elaboration of this group of compounds may afford novel antidiabetic agents.

Site‐Directed Mutations in the Third Intracellular Loop of the Serotonin 5‐HT1A Receptor Alter G Protein Coupling from Gi to Gs in a Ligand‐Dependent Manner

The effect of mutations (V344E and T343A/V344E) in the third intracellular loop of the serotonin 5-HT(1A) receptor expressed transiently in human embryonic kidney 293 cells have been examined in terms of receptor/G protein interaction and signaling. Serotonin, (R)-8-hydroxy-2-dipropylaminotetralin [(R)-8-OH-DPAT], and buspirone inhibited cyclic AMP production in cells expressing native and mutant 5-HT(1A) receptors. Serotonin, however, produced inverse bell-shaped cyclic AMP concentration-response curves at native and mutant 5-HT(1A) receptors, indicating coupling not only to G(i)/G(o), but also to G(s). (R)-8-OH-DPAT, however, induced stimulation of cyclic AMP production only after inactivation of G(i)/G(o) proteins by pertussis toxin and only at the mutant receptors. The partial agonist buspirone was unable to induce coupling to G(s) at any of the receptors, even after pertussis toxin treatment. The basal activities of native and mutant 5-HT(1A) receptors in suppressing cyclic AMP levels were not found to be significantly different. The receptor binding characteristics of the native and mutant receptors were investigated using the novel 5-HT(1A) receptor antagonist [(3)H]NAD-299. For other receptors, analogous mutations have produced constitutive activation. This does not occur for the 5-HT(1A) receptor, and for this receptor the mutations seem to alter receptor/G protein coupling, allowing ligand-dependent coupling of receptor to G(s) in addition to G(i)/G(o) proteins.

Muscarinic Receptor Stimulation Increases Regulators of G-protein Signaling 2 mRNA Levels through a Protein Kinase C-dependent Mechanism

RGS2, a member of the Regulators of G-protein Signaling family, modulates the activity of G-proteins coupled to the phosphoinositide signal transduction system, but little is known about what regulates RGS2. In human neuroblastoma SH-SY5Y cells stimulation of muscarinic receptors by carbachol activates phosphoinositide signaling and also caused a rapid, large, and long lasting increase in RGS2 mRNA levels. Direct activation of protein kinase C also rapidly increased RGS2 mRNA levels. Inhibition of protein kinase C with Ro31-8220, GF109203x, or Go6976 or down-regulation of protein kinase C inhibited increases in RGS2 mRNA levels induced by carbachol or by the activation of protein kinase C. Blockade of calcium signaling did not alter carbachol-induced increases in RGS2 mRNA levels. Neither activation of epidermal growth factor receptors nor stimulation of cyclic AMP production with forskolin increased RGS2 mRNA levels. Pretreatment with actinomycin D blocked increases in RGS2 mRNA levels but caused a surprisingly small, although statistically significant, partial blockade of protein kinase C-mediated feedback inhibition of carbachol-induced phosphoinositide hydrolysis. Thus, RGS2 mRNA levels are increased by activation of muscarinic receptors coupled to the phosphoinositide signal transduction system through a protein kinase C-dependent mechanism. This action may contribute to negative feedback control of this signaling cascade, but because the small contribution to negative feedback contrasts with the large and prolonged elevations in RGS2 mRNA levels, we speculate that its primary role may be in modulating other signaling components.

The Interaction of TSH Receptor Autoantibodies with 125I-Labelled TSH Receptor

Detergent-solubilized porcine TSH receptor (TSHR) has been labeled with 125I using a monoclonal antibody to the C-terminal domain of the receptor. The ability of sera containing TSHR autoantibody to immunoprecipitate the labeled receptor was then investigated. Sera negative for TSHR autoantibody (as judged by assays based on inhibition of labeled TSH binding to detergent-solubilized porcine TSHR) immunoprecipitated about 4% of the labeled receptor, whereas sera with high levels of receptor autoantibody immunoprecipitated more than 25% of the labeled receptor. The ability to immunoprecipitate labeled TSHR correlated well with ability of the sera to inhibit labeled TSH binding to the receptor (r = 0.92; n = 63), and this is consistent with TSHR autoantibodies in these samples being directed principally to a region of the receptor closely related to the TSH binding site. Preincubation of labeled TSHR with unlabeled TSH before reaction with test sera inhibited the immunoprecipitation reaction, providing further evidence for a close relationship between the TSHR autoantibody binding site(s) and the TSH binding site. This was the case whether the sera had TSH agonist (i.e., thyroid stimulating) or TSH antagonist (i.e., blocking) activities, thus, providing no clear evidence for different regions of the TSHR being involved in forming the binding site(s) for TSHR autoantibodies with stimulating and with blocking activities. The ability of TSHR autoantibodies to stimulate cyclic AMP production in isolated porcine thyroid cells was compared with their ability to immunoprecipitate labeled porcine TSHR. A significant correlation was observed (r = 0.58; n = 50; P < 0.001) and the correlation was improved when stimulation of cyclic AMP production was compared with inhibition of labeled TSH binding to porcine TSHR (r = 0.76). Overall, our results indicate that TSHR autoantibodies bind principally to a region on the TSHR closely related to the TSH binding site, and this seems to be the case whether the autoantibodies act as TSH agonists or antagonists.

Exploring the dynamics of regulation of G protein-coupled receptors using green fluorescent protein.

Exploring the dynamics of regulation of G protein-coupled receptors using green fluorescent protein.

Acute and chronic regulation of Na+/K+-ATPase transport activity in the RN22 Schwann cell line in response to stimulation of cyclic AMP production.

Na+/K+-ATPase-dependent Rb+ uptake of RN22 Schwann cells was stimulated by cholera toxin (0.25 microg/ml), forskolin (2 mM), or 8-bromo cAMP (1 mM). At 2 h Rb+ uptake was increased by 162+/-6% (cholera toxin), 151+/-14% (forskolin), and 207+/-15% (8-bromo cAMP). Cholera toxin or 8-bromo cAMP treatment for 12-24 h resulted in a second peak of Na+/K+-ATPase-dependent Rb+ transport activity of 186+/-12 and 265+/-9% of control, respectively. Cholera toxin also transiently stimulated the activity of the Na+, K+, 2Cl- -cotransporter with a peak at 2 h (179+/-9%), returning to basal levels by 24 h. Inhibition of the Na+,K+,2Cl- -cotransporter by bumetanide (0.1 mM) or by reduction of the Na+ gradient (10 mM veratridine treatment) prevented the early peak in ATPase activity but not the second peak. These results indicated that the early transient stimulation of Na+/K+ ATPase activity by cholera toxin was due to an increase in cellular Na+, secondary to stimulation of Na+,K+,2Cl -cotransport activity. Western blot analysis of cellular homogenates and purified membrane fractions showed that the second peak of Rb+ uptake activity was a result of translocation of transport protein from an intracellular microsomal pool to the plasma membrane. Rb+ uptake by dominant negative protein kinase A mutants of the RN22 cell was not stimulated by cholera toxin treatment (acute or chronic) confirming the cAMP/protein kinase A dependency of both acute and long-term regulation of transport activity. In the absence of a change in Michaelis constants or of an increase in total transport protein of cellular homogenates, neither a change in enzyme kinetics nor an increase in de novo synthesis of transport protein could account for the increase in transport activity.

Comparative studies on prostanoid receptors in human non-pigmented ciliary epithelial and mouse fibroblast cell lines

To examine the expression of functional prostanoid receptors in the human non-pigmented ciliary epithelial (ODMC1–2) and mouse fibroblast cell lines (NIH 3T3) we have measured the generation of the second messengers, cyclic AMP, inositol phosphates and the mobilization of intracellular calcium ([Ca 2+] i) following stimulation by prostaglandin receptor agonists. The amount of cyclic AMP generated was measured by a protein binding method. Radiolabeled inositol phosphates were separated using ion exchange columns and quantified by counting the radioactivity. For intracellular calcium measurements, Fura 2-AM loaded cells were stimulated by PG receptor agonists and the calcium activated fluorescence was measured in a spectrofluorometer. In the ODMC1–2 cell line, the formation of cyclic AMP was stimulated by prostaglandin E 2, butaprost and 11-deoxy-prostaglandin E 1. The stimulation of cyclic AMP production by prostaglandin E 2 was partially inhibited by the EP 4 receptor antagonist AH23848. Prostaglandin E 2 and 11-deoxy-prostaglandin E 1 stimulated the formation of cyclic AMP in NIH 3T3 cells. In ODMC1–2 cells, total inositol phosphate turnover was not increased by 17-phenyl-trinor-prostaglandin F 2α, 17-phenyl-trinor-prostaglandin E 2 or 11-deoxy-prostaglandin E 1. In contrast, all these agonists, with the exception of 11-deoxy-prostaglandin E 1, significantly increased total inositol phosphates in NIH 3T3 cells. In the NIH 3T3 cell line, only prostaglandin F 2α and 17-phenyl-trinor-prostaglandin F 2α increased [Ca 2+] i in a dose-dependent manner; in ODMC1–2 cells, neither these agonists nor 17-phenyl-trinor-prostaglandin E 2 increased [Ca 2+] i. The present studies suggest that in ODMC1–2 cells, EP 2 and EP 4 receptors but neither EP 1 nor FP receptors are expressed; these studies also imply, NIH 3T3 cells express EP 2 and FP receptors, while EP 1 receptors appear to be absent in this cell line.

The catecholaminergic stimulation of gonadotropin-releasing hormone release by GT 1-1 cells does not involve phosphoinositide hydrolysis

Gonadotropin-releasing hormone (GnRH) secretion is modulated by a large number of neuromediators, among which catecholamines play a central role. Previous results have shown that both dopamine (DA) and norepinephrine (NE) stimulate GnRH secretion in GT 1 neuronal cell lines. These stimulatory effects appear to involve D 1-dopaminergic and β 1-adrenergic receptors positively coupled to adenylate cyclase. However, in spite of a similar efficacy of these catecholamines to stimulate GnRH secretion, DA is two-fold more efficacious than NE to stimulate the formation of cyclic AMP. This rises the possibility that other signaling pathways and other receptor subtypes could be involved in the catecholaminergic stimulation of GnRH release. Since the signaling pathway triggered by phosphoinositide hydrolysis is a potent stimulator of GnRH secretion and appears to mediate the releasing actions of neuromediators such as histamine and endothelin, we investigated if this signaling pathway was also involved in the catecholaminergic stimulation of GnRH release in GT 1 cells. Both DA and NE stimulated inositol phosphates production in GT 1-1 cells with a very low potency and long latency with respect to GnRH secretion. Inositol phosphates production was stimulated by DA and NE only at a concentration of 100 μm, I.e. two to three orders of magnitude higher than the effective concentrations to maximally stimulate GnRH secretion. The effects of both catecholamines do not appear to be secondary to the stimulation of cyclic AMP production, since treatment of GT 1-1 cells with forskolin did not affect inositol phosphates production. The effects of DA and NE on inositol phosphates production were blocked by specific antagonists such as SCH-23390, spiroperidol and phentolamine. However, specific dopaminergic agonists such as SKF-38393 and bromocriptine, or adrenergic agonists such as clonidine, methoxamine and isoproterenol were not capable of stimulating inositol phosphates production. Thus, due to the low potency and apparent non-specificity of these effects, we conclude that inositol phosphates production is not involved in the catecholaminergic stimulation of GnRH release.

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates adenylyl cyclase and phospholipase C activity in rat cerebellar neuroblasts.

The presence of receptors for the novel neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) has been recently demonstrated in the external granule cell layer of the cerebellum, a germinative matrix that generates the majority of cerebellar interneurons. In the present study, we have taken advantage of the possibility of obtaining a culture preparation that is greatly enriched in immature cerebellar granule cells to investigate the effect of PACAP on the adenylyl cyclase and phospholipase C transduction pathways. The two molecular forms of PACAP, i.e., 27-(PACAP27) and 38-(PACAP38) amino-acid forms of PACAP, induced a dose-dependent stimulation of cyclic AMP production in granule cells. The potencies of PACAP27 and PACAP38 were similar (ED50 = 0.12 +/- 0.01 and 0.23 +/- 0.07 nM, respectively), whereas vasoactive intestinal polypeptide (VIP) was approximately 100 times less potent. PACAP27 and PACAP38 also induced a dose-dependent stimulation of polyphosphoinositide breakdown (ED50 = 19.1 +/- 6.3 and 13.4 +/- 6.0 nM, respectively), whereas VIP had no effect on polyphosphoinositide metabolism. The effect of PACAP38 on inositol phosphate formation was significantly reduced by U-73122 and by pertussis toxin, indicating that activation of PACAP receptors causes stimulation of a phospholipase C through a pertussis toxin-sensitive G protein. In contrast, forskolin and dibutyryl cyclic AMP did not affect PACAP-induced stimulation of inositol phosphates. Taken together, the present results demonstrate that PACAP stimulates independently the adenylyl cyclase and the phospholipase C transduction pathways in immature cerebellar granule cells. These data favor the concept that PACAP may play important roles in the control of proliferation and/or differentiation of cerebellar neuroblasts.

Cross reactivity studies of CRF-related peptides on insect Malpighian tubules

Manduca sexta diuretic peptide II (Mas-DPII) stimulates fluid secretion by adult Malpighian tubules and cyclic AMP production by larval proximal and adult tubules of M. sexta in a dose-dependent manner. Mas-DPII has no effect on fluid transport across the larval cryptonephric complex. M. sexta diuretic hormone (Mas-DH) and CRF-related insect diuretic peptides from Acheta domesticus, Locusta migratoria, and Periplaneta americana also cause similar increases in the production of cyclic AMP by the Malpighian tubules of both larval and adult M. sexta. Insect CRF-related diuretic peptides exhibit varying degrees of potency when assayed on Malpighian tubules from L. migratoria and A. domesticus. Sauvagine, bovine-CRF, and human-CRF have only a small, but significant, effect on cyclic AMP production by M. sexta Malpighian tubules. However, sauvagine, bovine-CRF, and sucker fish urotensin-I have no effect on L. migratoria tubules. Stimulation of cyclic AMP production by M. sexta Malpighian tubules could potentially be used as a screening assay to identify other insect CRF-related diuretic peptides.

Mobilization of intracellular Ca 2+ and stimulation of cyclic AMP production by κ opioid receptors expressed in Xenopus oocytes

The intracellular metabotropic pathway, following κ opioid receptor activation, was investigated in the Xenopus oocyte translation system. When oocytes were injected with cRNA for κ opioid receptor cDNA, U50488H rarely evoked phospholipase C-mediated, oscillatory Cl − current responses. However, after the oocytes were incubated with staurosporine, both the occurrence and the amplitude of U50488H-evoked responses were increased. The U50488H-evoked response was antagonized by naloxone and inhibited by pretreatment of the oocytes with pertussis toxin. When oocytes were coinjected with RNAs encoding κ opioid receptor and cystic fibrosis transmembrane conductance regulator (CFTR), treatment of the oocytes with forskolin and 3-isobutyl-1-methylxanthine (IBMX) evoked a smooth-shaped Cl − current flowing through the CFTR channels. The forskolin/IBMX-evoked response was never inhibited but was greatly potentiated in the presence of U50488H, indicating stimulation of adenylyl cyclase by U50488H. This U50488H-induced potentiation of CFTR channel opening was antagonized by naloxone and inhibited by pretreatment with pertussis toxin. These results suggest that κ opioid receptors mobilize intracellular Ca 2+ and stimulate cyclic AMP production by coupling positively to both phospholipase C and adenylyl cyclase via pertussis toxin-sensitive GTP-binding proteins in the oocytes.

Intracellular Ca 2+ transition assessed differentially among neocortical layers in brain slices during Ischemia-like condition

To examine the expression of functional prostanoid receptors in the human non-pigmented ciliary epithelial (ODMC1–2) and mouse fibroblast cell lines (NIH 3T3) we have measured the generation of the second messengers, cyclic AMP, inositol phosphates and the mobilization of intracellular calcium ([Ca2+]i) following stimulation by prostaglandin receptor agonists. The amount of cyclic AMP generated was measured by a protein binding method. Radiolabeled inositol phosphates were separated using ion exchange columns and quantified by counting the radioactivity. For intracellular calcium measurements, Fura 2-AM loaded cells were stimulated by PG receptor agonists and the calcium activated fluorescence was measured in a spectrofluorometer. In the ODMC1–2 cell line, the formation of cyclic AMP was stimulated by prostaglandin E2, butaprost and 11-deoxy-prostaglandin E1. The stimulation of cyclic AMP production by prostaglandin E2 was partially inhibited by the EP4 receptor antagonist AH23848. Prostaglandin E2 and 11-deoxy-prostaglandin E1 stimulated the formation of cyclic AMP in NIH 3T3 cells. In ODMC1–2 cells, total inositol phosphate turnover was not increased by 17-phenyl-trinor-prostaglandin F2α, 17-phenyl-trinor-prostaglandin E2 or 11-deoxy-prostaglandin E1. In contrast, all these agonists, with the exception of 11-deoxy-prostaglandin E1, significantly increased total inositol phosphates in NIH 3T3 cells. In the NIH 3T3 cell line, only prostaglandin F2α and 17-phenyl-trinor-prostaglandin F2α increased [Ca2+]i in a dose-dependent manner; in ODMC1–2 cells, neither these agonists nor 17-phenyl-trinor-prostaglandin E2 increased [Ca2+]i. The present studies suggest that in ODMC1–2 cells, EP2 and EP4 receptors but neither EP1 nor FP receptors are expressed; these studies also imply, NIH 3T3 cells express EP2 and FP receptors, while EP1 receptors appear to be absent in this cell line.

Regulation of Melanogenesis Induced by 5‐Methoxypsoralen Without Ultraviolet Light in Murine Melanoma Cells

Melanogenesis in melanoma cells can be enhanced by psoralens in the absence of UV light. Melanin biosynthesis is regulated by a number of melanocyte-specific proteins, including tyrosinase, DOPAchrome tautomerase (DCT), and tyrosinase-related protein-1 (TRP-1, gp75). To get more insight on the molecular mechanisms involved in psoralens-induced melanogenesis, we determined tyrosinase and DCT activities as well as mRNA and protein levels of tyrosinase, DCT, and TRP-1 in S91 mouse melanoma cells treated by 5-MOP. High concentration of 5-MOP (5 x 10(-5) M) induced a time-dependent increase of tyrosinase activity and melanin content, which was correlated to an increase of both mRNA and protein levels of tyrosinase. These results demonstrate that the 5-MOP stimulation of melanogenesis is related to increased tyrosinase synthesis. In addition, 5-MOP stimulated TRP-1 synthesis and induced a dose-dependent decrease of DCT activity without any modification in the expression of the protein. We explored then the signalling pathways involved in 5-MOP-induced melanogenesis and, particularly, the role of cyclic AMP and protein kinase C (PKC). A small stimulation of cyclic AMP production was observed in presence of 5-MOP. Furthermore, 1-oleoyl-2-acetylglycerol (OAG), a PKC activator, potentiated the 5-MOP stimulation of tyrosinase activity, while calphostin, a specific PKC inhibitor, inhibited the 5-MOP induction of tyrosinase activity. Phorbol-myristate acetate (PMA), described as a strong activator of PKC, inhibited also the effect of 5-MOP when used at long term. Taken together, these results demonstrate that in murine melanoma cells 5-MOP stimulates melanogenesis by increasing activity and synthesis of tyrosinase. Tyrosinase and TRP-1 expression are coordinately regulated by 5-MOP. Furthermore, a negative correlation between melanogenesis and DCT activity was observed under 5-MOP stimulation. At least, PKA and PKC systems appear to play an important role in the melanogenic effect of 5-MOP.

Expression of VIP receptors in mouse peritoneal macrophages: Functional and molecular characterization

Receptors for VIP in mouse peritoneal macrophages (MPM) were examined using [ 125I]labeled VIP as ligand. The receptor binding was rapid, reversible, saturable, specific, and dependent on time, pH, temperature and cell concentration. At 15°C, the stoichiometric data suggested the presence of two classes of VIP receptors with K d values of 1.05 ± 0.2 and 66.4 ±11.0 nM and binding capacities of 19.2 ± 2.8 and 706.6 ± 172.0 fmol VIP/10 6 cells. The interaction showed a high degree of specificity, as suggested by competition experiments with various peptides structurally related to VIP as follows: VIP > helodermin > rGRF > PHI ⪢ secretin. Glucagon, pancreastatin, somatostatin, insulin, and octapeptide of cholecystokinin (CCK 26–33) were ineffective at concentrations as high as 1 μM. VIP was a potent and efficient stimulator of cyclic AMP production in MPM. The stimulation was observed at a concentration as low as 0.01 nM VIP. Half-maximal stimulation (ED 50) was observed at 1.0 ± 0.2 nM VIP, and maximal stimulation (three-fold above basal levels) was obtained between 0.1 – 1 μM. The cyclic AMP system of mouse peritoneal macrophages showed a high specificity for VIP. The order of potency observed in inducing cyclic AMP production was VIP > helodermin > rGRF > PHI ⪢ secretin. Glucagon, insulin, pancreastatin, somatostatin and octapeptide of cholecystokinin did not modify cyclic AMP levels at concentrations as high as 1 μM. To characterize the molecular mass of VIP receptors, [ 125I]VIP was covalently bound to membranes from MPM using the cross-linker dithiobis(succinimidyl propionate) (DTSP); sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the solubilized membranes proteins revealed the presence of a major specific componentwith an M r value of 55 300 ± 1100 as estimated in denaturing conditions. Other proteins with M r values of 34 500 ± 700, 23 900 ± 500 and 18 500 ± 500 also were labeled. Taken together, these results demonstrate the presence of specific and functional VIP receptors in MPM and further support the concept of VIP as a neuroimmunoregulatory peptide.

Protein kinase C modulates parathyroid hormone- but not prostaglandin E2-mediated stimulation of cyclic AMP production via the inhibitory guanine nucleotide binding protein in UMR-106 osteosarcoma cells.

In UMR-106 osteosarcoma cells we found that PTH activated both the cAMP/protein kinase A and the Ca(2+)-dependent phosphoinositide/protein kinase C (PKC) pathways, but prostaglandin E2 (PGE2) activated only the cAMP pathway. Activation of PKC by the phorbol ester PMA had no effect on cAMP production but enhanced PTH-stimulated cAMP production by 50% or more; the effect on PGE2-induced cAMP was negligible. Inhibition of the alpha-subunit of the inhibitory guanine nucleotide binding protein (Gi) by pertussis toxin pretreatment also enhanced PTH-mediated cAMP production but had no effect on PGE2-induced cAMP production. These results suggest that although PTH-mediated adenylate cyclase activity is regulated via both the stimulatory (Gs) and inhibitory (Gi) guanine nucleotide binding proteins, only Gs regulates PGE2-mediated adenylate cyclase activity in UMR-106 cells. Costimulation with pertussis toxin and PMA did not increase PTH-stimulated cAMP production above that obtained with PMA alone. This implies a similar target of action for pertussis toxin and PMA, that is, the alpha-subunit of Gi. The alpha-subunit of Gi was found to be a substrate for in vitro PKC phosphorylation of membrane fractions from UMR-106 cells, seen as a +/- 40 kD band on SDS-PAGE. Stimulation of in situ 32P-labeled cells with either PMA or PTH also enhanced incorporation of 32P into the 40 kD band. Using the peptide antisera AS/7 and EC/2, we showed that pertussis toxin-labeled subunits of both Gi1 alpha/Gi2 alpha and Gi3 alpha could be immunoprecipitated, respectively, but immunoprecipitation of membrane proteins after in situ phosphorylation and stimulation with PMA precipitated only Gi2 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)