Basal Accumulation Research Articles

Agonist and inverse agonist actions of beta-blockers at the human beta 2-adrenoceptor provide evidence for agonist-directed signaling.

Beta-blockers have beneficial effects in heart failure, although the underlying mechanism is unknown. Beta2-adrenoceptors, however, are proportionally higher in the failing human heart. This study shows several clinically used beta-blockers are agonists at the human beta2-adrenoceptor. Although these agonist effects were small at the cAMP level, they were substantial at the level of cAMP response element (CRE)-mediated gene transcription. Some of the effects of "beta-blockers" seen in heart failure may be related to the beta2-agonist actions of these compounds. CRE-gene transcription responses to beta2-agonists, forskolin, and cAMP-analogs were sensitive to p42/44-mitogen-activated protein (MAP) kinase pathway inhibitors. p42/44-MAP kinase activation was also shown directly by western blotting and enzyme-linked immunosorbent assay techniques. N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89; a protein kinase A inhibitor) stimulated cAMP accumulation and CRE gene transcription via the beta2-adrenoceptor at concentrations at which protein kinase A was inhibited, providing evidence for an alternative pathway. Propranolol, however, produced paradoxical effects; it reduced basal cAMP accumulation (via beta2-mediated inverse agonism) but stimulated beta2-mediated CRE gene transcription. This cannot be explained by a sequential pathway from Gs-adenylyl cyclase-cAMP to CRE binding protein phosphorylation. Both responses to propranolol were insensitive to pertussis toxin, thus excluding Gi-protein involvement. Propranolol CRE gene transcription responses were attenuated by p42/44-MAP kinase inhibitors and propranolol was also found to directly stimulate the p42/44-MAP kinase pathway. Studies of inositol phosphate accumulation and of protein kinase C or Rho kinase inhibitors on CRE-gene transcription provided no evidence for Gq/11 or G12/13 involvement. These data suggest that propranolol can simultaneously act as an inverse agonist through a Gs-coupled mechanism while stimulating the p42/44-MAP kinase pathway through an alternative G-protein-independent mechanism.

Involvement of K +channels and calcium-dependent pathways in the action of T 3 on amino acid accumulation and membrane potential in Sertoli cells of immature rat testis

The purpose of this study was to investigate the involvement of calcium in K + currents and its effects on amino acid accumulation and on the membrane potential regulated by tri-iodo-L-thyronine (T 3) in Sertoli cells. Immature rat testes were pre-incubated for 30 min in Krebs-Ringer bicarbonate buffer and incubated for 60 min in the presence of [ 14C]methylaminoisobutyric acid with and without T 3 or T 4 (dose-response curve). Specific channel blockers or chelating agents were added at different concentrations during pre-incubation and incubation periods to study the basal amino acid accumulation and a selected concentration of each drug was chosen to analyze the influence on the stimulatory hormone action. All amino acid accumulation experiments were carried out in a Dubnoff metabolic incubator at 32 °C, pH 7.4 and gassed with O 2:CO 2 (95:5; v/v). Seminiferous tubules from immature Sertoli cell-enriched testes were used for the electrophysiology experiments. Intracellular recording of the Sertoli cells was carried out in a chamber perfused with KRb with/without T 3, T 4 or blockers and the membrane potential was monitored. We found that T 3 and T 4 stimulated α-[1- 14C] methylaminoisobutyric acid accumulation in immature rat testes and induced a membrane hyperpolarization in Sertoli cells. The action of T 3 on amino acid accumulation and on the hyperpolarizing effect was inhibited by the K +-ATP channel blocker tolbutamide as well as the voltage-dependent Ca 2+ channel blocker verapamil. These results clearly demonstrate for the first time the existence of an ionic mechanism related to Ca 2+ and K + fluxes in the rapid, nongenomic action of T 3.

All-trans retinoic acid- and N-(4-hydroxyphenil)-retinamide-induced growth arrest and apoptosis in orbital fibroblasts in Graves’ disease

In this study, we evaluated by reverse transcription-polymerase chain reaction (RT-PCR) the expression pattern of retinoic acid receptors (RAR) α, β, and γ and cellular retinoic acid binding protein-I (CRBP-I) genes in 12 primary cultures of fibroblasts (F) from orbital tissue of Graves’ ophthalmopathy (GO) patients. We also studied the in vitro effects of all-trans retinoic acid (RA) and N-(4-hydroxyphenil)-retinamide (4HPR), a less toxic and better tolerated synthetic derivative of RA, on cell morphology, growth, apoptosis, and cyclic adenosine monophosphate (cAMP) accumulation. All primary cultures expressed RAR α, β, γ, and CRBP-I. FGO treated with RA and 4HPR (10−7 mol/L) presented morphologic changes and significantly inhibited cell growth after 72 hours. At 96 hours of drug exposure, apoptosis was detected in 15% and 50% of RA- and 4HPR (10−7 mol/L)-treated cells, and p53 protein increased in cell lysates. 4HPR induced a 70% decrease of Bcl-2 protein. After 30 minutes of RA and 4HPR (10−7 mol/L) exposure, a 20% decrease of basal cAMP accumulation was seen, and forskolin cAMP-induced increase was abolished. The expression of RAR α, β, γ, and CRBP-I in primary cultures of FGO indicates that they are targets for retinoids. Moreover, we show that RA and 4HPR are able to induce morphologic changes, inhibition of cell growth, and apoptosis in FGO exerting their effects through RAR-modulated pathways. The rapid inhibition of cAMP accumulation indicates that a novel nonclassic retinoid pathway may also be involved. Finally, the potent in vitro effects of 4HPR, a retinoid derivative with fewer adverse reactions in vivo, could justify further investigations on a clinical application of retinoids in GO.

The third intracellular loop and the carboxyl terminus of beta2-adrenergic receptor confer spontaneous activity of the receptor.

It is well established that the beta2-adrenergic receptor (beta2-AR) exhibits a robust ligand-independent activity, whereas this property is considerably weaker in the closely related beta1-AR subtype. To identify the potential domain(s) of beta2-AR responsible for the spontaneous receptor activation, we created three chimeras in which the third intracellular loop (beta1/beta2-Li3) or the carboxyl terminus (beta1/beta2-CT) or both domains (beta1/beta2-Li3CT) of beta1-AR are replaced by the corresponding parts of the beta2-AR. Using adenoviral gene transfer, we individually expressed these beta1/beta2-AR chimeras in mouse cardiomyocytes lacking both native beta1-AR and beta2-AR (beta1/beta2 double knockout), and examined their possible spontaneous activities. Overexpression of these beta1/beta2-AR chimeras markedly elevated basal cAMP accumulation and myocyte contractility in the absence of agonist stimulation compared with those infected by a control adenovirus expressing beta-galactosidase or an adenovirus expressing wild type beta1-AR. These effects were fully reversed by a beta2-AR inverse agonist, (+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol (ICI 118,551; 5 x 10-7 M), regardless of inhibition of Gi with pertussis toxin, but not by a panel of beta1-AR antagonists, including [2-(3-carbamoyl-4-hydroxyphenoxy)-ethylamino]-3-[4-(1-methyl-4-trifluormethyl-2-imidazolyl)-phenoxy]-2-propanolmethanesulfonate (CGP20712A), betaxolol, bisoprolol, and metoprolol. Furthermore, we have shown that the C-terminal postsynaptic density 95/disc-large/ZO-1 (PDZ) motif of beta1-AR is not responsible for the lack of beta1-AR spontaneous activation, although it has been known that the beta1-AR PDZ motif prevents the receptor from undergoing agonist-induced trafficking and Gi coupling in cardiomyocytes. Taken together, the present results indicate that both the third intracellular loop and the C terminus are involved in beta2-AR spontaneous activation and that either domain seems to be sufficient to confer the receptor spontaneous activity.

ScFv Single Chain Antibody Variable Fragment as Inverse Agonist of the β2-Adrenergic Receptor

Antibodies directed against the second extracellular loop of G protein-coupled receptors were shown to possess functional activities. Using a functional monoclonal antibody against the human beta2-adrenergic receptor, a scFv fragment with high affinity for the target epitope was constructed and produced. The fragment recognized the beta2-adrenergic receptors on A431 cells, blocked cAMP accumulation induced by the beta2-agonist salbutamol, and decreased basal cAMP accumulation in the same cells. Their in vitro activity was tested on neonatal rat cardiomyocytes. The antibody fragments blocked the chronotropic activity induced by the beta2-agonist clenbuterol. They also decreased the in vivo heart beating frequency of mice pretreated with bisoprolol (a beta1-adrenergic receptor antagonist) for 4 min after injection. The immunological approach presented here may serve as a strategy for the synthesis of a new class of allosteric modulators for G protein-coupled receptors.

Lidocaine attenuates muscarinic receptor-mediated inhibition of adenylyl cyclase in airway smooth muscle

We examined how lidocaine affects muscarinic receptor-mediated inhibition of adenylyl cyclase in bovine tracheal smooth muscles. Lidocaine (100 μM) augmented the relaxant responses to forskolin in the bovine tracheal smooth muscle contracted with methacholine (0.3 μM). On the other hand, lidocaine failed to affect the relaxant effects of forskolin on the histamine (100 μM)- and KCl (40 mM)-contracted preparations. Lidocaine (100 μM) enhanced both basal and forskolin-stimulated cAMP accumulation in the presence of methacholine (0.3 μM). However, in the absence of methacholine, neither basal nor forskolin-stimulated cAMP accumulation was affected by lidocaine. Similar phenomenon was observed when the bovine tracheal smooth muscles were treated with methoctramine (0.03 μM). In radioligand binding experiments, lidocaine inhibited [ 3H] N-methyl scopolamine binding to cloned human muscarinic receptors (M 1–M 5) expressed in Chinese hamster ovary cells. These results suggest that lidocaine prevents muscarinic receptor-mediated signaling pathway and thereby reverses inhibition of adenylyl cyclase by methacholine in bovine tracheal smooth muscle.

Peptide and nonpeptide antagonist interaction with constitutively active human AT 1 receptors

Wild type human AT 1 receptors (WT-AT 1) and mutant receptors, in which Asn 111 was replaced by glycine (N111G), alanine (N111A) and serine (N111S), or in which Asp 281 was replaced by alanine (D281A) or in which N111G and D281A replacements were combined, were transiently expressed in CHO-K1 cells. While the biphenyltetrazole compound candesartan dissociated slowly and behaved as an insurmountable antagonist for WT-AT 1, it dissociated swiftly and only produced a rightward shift of the angiotensin Ang II- and -IV dose–response curves for inositol phosphate (IP) accumulation in cells expressing N111G. [ 3 H ]candesartan competition binding yielded the same potency order of the related biphenyltetrazoles for WT-AT 1 and mutated receptors, i.e. candesartan>EXP3174>irbesartan>losartan. Affinities were equal for WT-AT 1 and D281A and 40- to 400-fold lower for all Asn 111 mutants. Mutations did not affect the affinity of the peptide antagonist [Sar 1Ile 8]Ang II (SARILE). Basal IP accumulation in cells with WT-AT 1 was not affected by any biphenyltetrazole antagonists and was increased by SARILE to 19% of the maximal Ang II stimulation. Basal IP accumulation was higher for cells expressing the Asn 111-mutated receptors. For N111G, this accumulation was partially inhibited by all the biphenyltetrazoles upon long-term (18 hr) exposure. In these cells SARILE produced the same maximal stimulation as Ang II. Asn 111-mutated AT 1 receptors are thought to mimic the pre-activated state of the wild type receptor and comparing the efficacy and affinity of ligands for such mutated receptors facilitate the distinction of partial (SARILE) and inverse (biphenyltetrazoles) agonists from true antagonists.

The Adipocyte Plasma Membrane Caveolin Functional/Structural Organization Is Necessary for the Efficient Endocytosis of GLUT4

It is well established that insulin stimulation of glucose uptake requires the translocation of intracellular localized GLUT4 protein to the cell surface membrane. This plasma membrane-redistributed GLUT4 protein was partially co-localized with caveolin as determined by confocal fluorescent microscopy but was fully excluded from lipid rafts based upon Triton X-100 extractability. Cholesterol depletion with methyl-beta-cyclodextrin, filipin, or cholesterol oxidase resulted in an insulin-independent increase in the amount of plasma membrane-localized GLUT4 that was fully reversible by cholesterol replenishment. This basal accumulation of cell surface GLUT4 occurred due to an inhibition of GLUT4 endocytosis. However, this effect was not specific since cholesterol extraction also resulted in a dramatic inhibition of clathrin-mediated endocytosis as assessed by transferrin receptor internalization. To functionally distinguish between caveolin- and clathrin-dependent endocytic processes, we took advantage of a dominant-interfering caveolin 1 mutant (Cav1/S80E) that specifically disrupts caveolae organization. Expression of Cav1/S80E, but not the wild type (Cav1/WT) or Cav1/S80A mutant, inhibited cholera toxin B internalization without any significant effect on transferrin receptor endocytosis. In parallel, Cav1/S80E expression increased the amount of plasma membrane-localized GLUT4 protein in an insulin-independent manner. Although Cav1/S80E also decreased GLUT4 endocytosis, the extent of GLUT4 internalization was only partially reduced ( approximately 40%). In addition, expression of Cav1/WT and Cav1/S80A enhanced GLUT4 endocytosis by approximately 20%. Together, these data indicate that the endocytosis of GLUT4 requires clathrin-mediated endocytosis but that the higher order structural organization of plasma membrane caveolin has a significant influence on this process.

Impaired desensitization of a mutant adrenocorticotropin receptor associated with apparent constitutive activity.

A naturally occurring ACTH receptor [melanocortin 2 receptor (MC2R)] mutation (F278C) has been identified in a subject with ACTH-independent Cushing's syndrome. Functional characterization of this mutant receptor reveals that it is associated with elevated basal cAMP accumulation when compared with wild-type receptor-expressing cell lines. Dose responsiveness is similar between wild-type and mutant receptors in cell lines expressing similar numbers of binding sites. In view of the location of this mutation in the C-terminal tail of the MC2R, desensitization and internalization were investigated and found to be impaired. Inhibition of protein kinase A by H89 blocks wild-type MC2R desensitization and also results in increased basal activity, as does alanine substitution of Ser 280 in the C-terminal tail. Alanine substitution of Ser 208, the consensus protein kinase A phosphorylation target in the third cytoplasmic loop also results in a reduction in desensitization without significant change in basal activity or internalization. These findings suggest a novel mechanism is involved in the apparently constitutive activation of the MC2R in which failure of desensitization appears to be associated with enhanced basal receptor activity.

The East Bull Lake Intrusive Suite: Remnants of a 2.48 GaLarge Igneous and Metallogenic Province in the Sudbury Area of the CanadianShield

The 2.49 to 2.475 Ga intrusions of the East Bull Lake intrusive suite occur in an east-northeast-trending discontinuous belt along the presently exposed boundary between the Archean Superior and the Proterozoic Southern provinces of the Canadian Shield near Sudbury, Ontario. The East Bull Lake intrusive suite is part of a regional Paleoproterozoic magmatic event, extending from 2.49 to 2.44 Ga, which also includes bimodal volcanic rocks, felsic plutons, and the regionally extensive Hearst and Matachewan dike swarms. This regional magmatic event is thought to be the result of a mantle plume-driven, intracontinental rifting event that led to the development of a major basin to the south that was subsequently filled by sedimentary rocks of the Huronian Supergroup. The East Bull Lake intrusive suite appears to have been emplaced along a major axial-rift fault related to this rifting event. Compelling field and geochemical evidence summarized here indicates that the three largest East Bull Lake suite intrusions, the East Bull Lake, Agnew Lake, and River Valley intrusions, crystallized from similar, low Ti, high Al tholeiitic parent magmas that originated in deeper, more basic chambers. It is proposed that the primary magmas for the East Bull Lake suite intrusions were second-stage melts derived from a sublithospheric depleted mantle source that had been modified by Neoarchean subduction and accretion events. Most of the petrological characteristics of the East Bull Lake suite intrusions reflect plagioclase-dominated fractional crystallization that generated a pronounced Fe enrichment trend in the residual magmas. Orthopyroxene is the high-temperature pyroxene and occurs as a cumulus phase through most of the stratigraphy of each intrusion of the suite. Local olivine-rich cumulates, present in all of the major East Bull Lake suite plutons, in some instances appear to be comagmatic with associated plagioclase-rich cumulates. A distinctive marginal facies, comprising brecciated and locally thermally recrystallized and/or partially melted footwall rocks, is a common feature of the East Bull Lake suite plutons and indicates that a high-energy flow regime existed during the initial stages of emplacement. This marginal unit typically grades into a heterolithic, inclusion-rich gabbronorite that contains erratically distributed mafic autoliths and footwall-derived xenoliths. Chemical contamination of the mineralized matrix of these inclusion- bearing rocks can be extreme at a local scale. The inclusion-rich zone is overlain by a thick interval of undifferentiated, plagioclase-rich cumulates that locally display spectacular glomerocrystic textures. Rhythmically and irregularly, modally layered leucogabbronorite and gabbronorite make up much of the middle parts of the stratigraphy. Massive to crudely layered ferrogabbro and ferrosyenite only occur in the uppermost part of the Agnew Lake intrusion. Varitextured gabbro and pegmatitic gabbro occur as irregular layers and local veins and pods throughout the stratigraphy. The East Bull Lake suite is currently being explored for contact-type platinum-group element (PGE)-Cu-Ni mineralization that has a clear spatial association with orthopyroxene-rich cumulates which are otherwise poorly represented in these intrusions. Disseminated PGE- and Cu-rich sulfide mineralization is concentrated within the inclusion-bearing zones but extends upward into overlying plagioclase cumulates and appears to have collected along the lower margins of these intrusions through the combined action of vigorous convection of the resident magma and downward percolation of dense, mafic residual liquid. The resultant narrow zones of sulfide mineralization were thus derived from a much larger volume of parent magma. Feeder dikes to the mineralized parts of the intrusions appear to have been saturated in sulfide upon emplacement and had relatively high background PGE contents compared to those for other known magmatic PGE deposits; however, the economic potential of the contact-type mineralization hinges on the efficacy of the physical concentration process, which upgraded the metal content and size of the original sulfide particles inside the magma chambers. Based on the proposed basal accumulation model, the best prospective mineralization is most likely to occur in embayments along the sidewall and floor of the intrusions.

Glycoxidized low-density lipoprotein downregulates endothelial nitricoxide synthase in human coronary cells

ObjectivesWe examined the hypothesis that low-density lipoprotein (LDL) that is both oxidized and glycosylated potently downregulates the expression of endothelial nitric oxide synthase III (NOSIII) in human coronary endothelial cells. BackgroundDiabetes mellitus is accompanied by both oxidation and glycosylation of LDL, but the potential interaction of these processes or the pathophysiologic effects of these modified lipoproteins on arteries are poorly understood. MethodsLow-density lipoprotein was glycoxidized in vitro, and Western and Northern blot analyses were used to investigate NOSIII expression in human coronary endothelial cells. Nitric oxide (NO) bioactivity was represented by both basal and bradykinin-stimulated cellular cyclic guanosine monophosphate accumulation and l-citrulline conversion from l-arginine. Nuclear run-on experiments were performed to study the transcription rate of nascent NOSIII messenger ribonucleic acid (mRNA). ResultsData showed a significant decrease in NOSIII expression after 24-h treatment with glycosylated low-density lipoprotein (glycLDL) and oxidized low-density lipoprotein (oxLDL). Accordingly, we observed a significant dose-dependent reduction in NO bioactivity (p < 0.05 to p < 0.001 vs. untreated cells, native low density lipoprotein [nLDL], glycLDL, and oxLDL). Glyc-oxLDL did not reduce the half-life of NOSIII mRNA or significantly enhance l-citrulline conversion. Nuclear run-on experiments showed that high doses of glyc-oxLDL can reduce the transcription rate of nascent NOSIII mRNA (densitometric analysis revealed a reduction of 25% [p < 0.05 vs. untreated cells, nLDL, and glycLDL] after treatment of cells with 300 μg/ml glyc-oxLDL). The effects of glyc-oxLDL are not related to the higher levels of oxidative compounds in comparison to those of oxLDL. ConclusionsThese results indicate that glyc-oxLDL, per se, may influence signal transduction pathways involving NO-mediated regulatory signals and NOSIII activity in human endothelial cells. This phenomenon can adversely influence the evolution of clinical vascular complications, coronary heart disease, and atherogenesis in diabetic patients.

Enhanced calcium signaling and acid secretion in parietal cells isolated from gastrin-deficient mice.

Gastrin-deficient mice have impaired basal and agonist-stimulated gastric acid secretion. To analyze whether an intrinsic parietal cell defect contributed to the reduced acid secretion, we analyzed parietal cell calcium responses and acid secretory function in vitro. Parietal cells were purified by light-scatter cell sorting and calcium responses to gastrin, histamine, and carbachol were measured in gastrin-deficient and wild-type mice cell preparations. Surprisingly, basal and histamine-induced calcium concentrations were higher in the mutant cell preparations. [(14)C]aminopyrine uptake analysis in acutely isolated gastric glands revealed that basal acid accumulation was enhanced in gastrin-deficient cell preparations as well as on treatment with carbachol or histamine. These results suggested that an intrinsic parietal cell defect was not responsible for the reduced acid secretion in gastrin-deficient mice. Flow cytometric analysis of dispersed, H(+)-K(+)-ATPase-immunostained gastric mucosal preparations revealed a marked increase in parietal cell number in gastrin-deficient mice, which may have accounted for the enhanced in vitro acid secretion detected in this study. Parietal cells were found to be significantly smaller in the mutant cell preparations, suggesting that gastrin stimulation modulates parietal cell morphology.

Quantitative trait loci affecting initial sensitivity and acute functional tolerance to ethanol-induced ataxia and brain cAMP signaling in BXD recombinant inbred mice.

In previous work, we identified genetic correlations between cAMP accumulation in the cerebellum and sensitivity to the incoordinating effects of ethanol. A genetic correlation suggests that common genes underlie the phenotypes investigated. One method for provisionally identifying genes involved in a given phenotypic measure is quantitative trait locus (QTL) analysis. Using a panel of 30 BXD recombinant inbred strains of mice and the progenitors (DBA/2J and C57BL/6J), and the dowel test for ataxia, we measured the blood ethanol concentrations at the time an animal first fell from the dowel and acute functional tolerance (AFT), and investigated cAMP signaling in the cerebellum. Cyclic AMP accumulation was measured in whole-cell preparations of cerebellar minces from individual mice under basal or stimulated conditions. We conducted a genome-wide QTL analysis of the behavioral and biochemical measures with >2000 genetic markers to identify significant associations. Western blot and comparative sequencing analysis were used to compare cAMP response element binding protein (CREB) levels and protein-coding sequence, respectively. QTL analyses correlating strain means with allelic status at genetic markers identified several significant associations (p < 0.01). Analysis of variance revealed an effect of strain on behavioral and biochemical measures. There was a significant genetic correlation between initial sensitivity and basal cAMP accumulation in the cerebellum. We identified 6 provisional QTLs for initial sensitivity on four chromosomes, 6 provisional QTLs for AFT on four chromosomes, and 11 provisional QTLs for cAMP signaling on nine chromosomes. Two loci were found to overlap for measures of initial sensitivity and for cAMP signaling. Given the genetic correlation between initial sensitivity and basal cAMP accumulation, we investigated candidate genes in a QTL on chromosome 1. Comparative sequence analysis was performed, and protein levels were compared between C57 and DBA mice for Creb1. No significant differences were detected in coding sequence or protein levels for CREB. These results suggest that although ethanol sensitivity and cAMP signaling are determined by multiple genes, they may share certain genetic codetermination.

Aging alters light- and PACAP-induced cAMP accumulation in the suprachiasmatic nucleus of female rats

Light-induced release of pituitary adenylate cyclase activating peptide (PACAP) from retinal ganglion cells can modulate the phase-shifting effects of light though a cAMP-mediated mechanism in neurons of the suprachiasmatic nucleus (SCN). Since older animals (12 months or older) show a reduced behavioral and cellular response to light presented during the early portion of the dark phase of the cycle, we hypothesized that aging may alter the ability of PACAP and cAMP to modulate the phase shifting effects of light. In Expt. 1, we examined basal and light-induced cAMP accumulation at zeitgeber time 14 (ZT14 where ZT0 is the time of lights on). Light exposure resulted in a significant increase in cAMP accumulation in SCN tissue collected from young, but not middle-aged animals. The failure to see an increase in cAMP accumulation in the SCN of middle-aged animals may be related to the fact that basal levels of cAMP were elevated in the SCN of these animals at ZT14. In Expt. 2, we used an in vitro slice preparation of the SCN to determine if aging altered the ability of PACAP to stimulate cAMP accumulation in the SCN at ZT14. PACAP stimulated cAMP in the SCN of both young and middle-aged animals. However, PACAP-induced cAMP accumulation was lower in the SCN of middle-aged animals. Based on these results, we conclude that age-related changes in the responsiveness of the SCN to light input are due to: (1) changes in other input pathways capable of modulating cAMP, and (2) decreases in PACAP receptors in SCN neurons.

A fusion protein of the human P2Y(1) receptor and NTPDase1 exhibits functional activities of the native receptor and ectoenzyme and reduced signaling responses to endogenously released nucleotides.

To begin to address the functional interactions between constitutively released nucleotides, ectonucleotidase activity, and P2Y receptor-promoted signaling responses, we engineered the human P2Y(1) receptor in a fusion protein with a member of the ectonucleoside triphosphate diphosphohydrolase family, NTPDase1. Membranes prepared from Chinese hamster ovary (CHO)-K1 cells stably expressing either wild-type NTPDase1 or the P2Y(1) receptor-NTPDase1 fusion protein exhibited nucleotide-hydrolytic activities that were over 300-fold greater than activity measured in membranes from empty vector-transfected cells. The molecular ratio for nucleoside triphosphate versus diphosphate hydrolysis was approximately 1:0.4 for both the wild-type NTPDase1 and P2Y(1)-NTPDase1 fusion protein. Stable expression of the P2Y(1)-NTPDase1 fusion protein conferred an ADP and 2MeSADP-promoted Ca(2+) response to CHO-K1 cells. Moreover, the maximal capacity of the nonhydrolyzable agonist ADPbetaS to stimulate inositol phosphate accumulation was similar, and the EC(50) of ADPbetaS was lower in the fusion protein than the wild-type receptor. In contrast, the substantial nucleotide-hydrolyzing activity of the fusion protein resulted in a greater than 50-fold shift to the right of the concentration-effect curve of ADP for activation of phospholipase C compared with the wild-type receptor. Heterologous expression of the P2Y(1) and other P2Y receptors results in marked increases in basal inositol phosphate levels. Given the high nucleotidase activity and apparently normal receptor signaling activity of the P2Y(1) receptor-NTPDase1 fusion protein, we quantitated basal inositol phosphate accumulation in cells stably expressing either the wild-type P2Y(1) receptor or the fusion protein. Although marked elevation of inositol phosphate levels occurred with wild-type P2Y(1) receptor expression, levels in cells expressing the fusion protein were not different from those in wild-type CHO-K1 cells.

Expression of G(alpha)(s) proteins and TSH receptor signalling in hyperfunctioning thyroid nodules with TSH receptor mutations.

Constitutively activating mutations of the thyrotrophin receptor (TSHR) are the main molecular cause of hyperfunctioning thyroid nodules (HTNs). The G protein coupling is an important and critical step in the TSHR signalling which mainly includes G(alpha)(s), G(alpha)(i) and G(alpha)(q)/11 proteins. We investigated the in vitro consequences of overexpressing G(alpha) proteins on signalling of the wild-type (WT) or mutated TSHR. Moreover, we investigated whether changes in G(alpha) protein expression are pathophysiologically relevant in HTNs or cold thyroid nodules (CTNs). Wild-type TSH receptor and mutated TSH receptors were coexpressed with G(alpha)(s), G(alpha)(i) or G(alpha)(q)/11, and cAMP and inositol phosphate (IP) production was measured after stimulation with TSH. The expression of G(alpha)(s), G(alpha)(i) and G(alpha)(q)/11 proteins was examined by Western blotting in 28 HTNs and 14 CTNs. Coexpression of G(alpha)(s) with the WT TSH receptor in COS 7 cells significantly increased the basal and TSH-stimulated cAMP accumulation while coexpression of the G(alpha)(q) or G(alpha)11 protein significantly increased the production of cAMP and inositol triphosphate (IP(3)). The coexpression of the TSH receptor mutants (I486F, DEL613-621), known to couple constitutively to G(alpha)(s) and G(alpha)(q) with G(alpha)(s) and G(alpha)(q)/11, significantly increased the basal and stimulated cAMP and IP(3) accumulation. Coexpression of the TSH receptor mutant V556F with G(alpha)(s) only increased the basal and stimulated cAMP production while its coexpression with G(alpha)(q)/11 increased the basal and stimulated IP(3) signalling. The expression of G(alpha)(s) protein subunits determined by Western blotting was significantly decreased in 14 HTNs with a constitutively activating TSH receptor mutation in comparison with the corresponding surrounding tissue, while in 14 HTNs without TSH receptor or G(alpha)(s) protein mutation and in 14 CTNs the expression of G(alpha)(s) protein was not different compared with the surrounding tissue. The expression of G(alpha)(i) and G(alpha)(q)/11 proteins in HTNs or CTNs was not significantly different compared with the surrounding tissue. The reduced expression of G(alpha)(s) protein subunits in HTNs with TSHR mutations could act as a feedback mechanism to desensitise the chronically stimulated cAMP cascade. As G(alpha) protein expression was not significantly increased in the majority of CTNs and HTNs an influence of G(alpha) overexpression on TSH signalling could be excluded in these nodules.

Calcium-independent and cAMP-dependent Modulation of Soluble Guanylyl Cyclase Activity by G Protein-coupled Receptors in Pituitary Cells

It is well established that G protein-coupled receptors stimulate nitric oxide-sensitive soluble guanylyl cyclase by increasing intracellular Ca(2+) and activating Ca(2+)-dependent nitric-oxide synthases. In pituitary cells receptors that stimulated adenylyl cyclase, growth hormone-releasing hormone, corticotropin-releasing factor, and thyrotropin-releasing hormone also stimulated calcium signaling and increased cGMP levels, whereas receptors that inhibited adenylyl cyclase, endothelin-A, and dopamine-2 also inhibited spontaneous calcium transients and decreased cGMP levels. However, receptor-controlled up- and down-regulation of cyclic nucleotide accumulation was not blocked by abolition of Ca(2+) signaling, suggesting that cAMP production affects cGMP accumulation. Agonist-induced cGMP accumulation was observed in cells incubated in the presence of various phosphodiesterase and soluble guanylyl cyclase inhibitors, confirming that G(s)-coupled receptors stimulated de novo cGMP production. Furthermore, cholera toxin (an activator of G(s)), forskolin (an activator of adenylyl cyclase), and 8-Br-cAMP (a permeable cAMP analog) mimicked the stimulatory action of G(s)-coupled receptors on cGMP production. Basal, agonist-, cholera toxin-, and forskolin-stimulated cGMP production, but not cAMP production, was significantly reduced in cells treated with H89, a protein kinase A inhibitor. These results indicate that coupling seven plasma membrane-domain receptors to an adenylyl cyclase signaling pathway provides an additional calcium-independent and cAMP-dependent mechanism for modulating soluble guanylyl cyclase activity in pituitary cells.

Inhibition of adenylyl cyclase by neuronal P2Y receptors.

P2Y receptors inhibiting adenylyl cyclase have been found in blood platelets, glioma cells, and endothelial cells. In platelets and glioma cells, these receptors were identified as P2Y(12). Here, we have used PC12 cells to search for adenylyl cyclase inhibiting P2Y receptors in a neuronal cellular environment. ADP and ATP (0.1 - 100 microM) left basal cyclic AMP accumulation unaltered, but reduced cyclic AMP synthesis stimulated by activation of endogenous A(2A) or recombinant beta(2) receptors. Forskolin-dependent cyclic AMP production was reduced by <or=1 microM and enhanced by 10 - 100 microM ADP; this latter effect was turned into an inhibition when A(2A) receptors were blocked. The nucleotide inhibition of cyclic AMP synthesis was not altered when P2X receptors were blocked, but abolished by pertussis toxin. The rank order of agonist potencies for the reduction of cyclic AMP was (IC(50) values): 2-methylthio-ADP (0.12 nM)=2-methylthio-ATP (0.13 nM)>ADPbetaS (71 nM)>ATP (164 nM)=ADP (244 nM). The inhibition by ADP was not antagonized by suramin, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid, or adenosine-3'-phosphate-5'-phosphate, but attenuated by reactive blue 2, ATP(alpha)S, and 2-methylthio-AMP. RT - PCR demonstrated the expression of P2Y(2), P2Y(4), P2Y(6), and P2Y(12), but not P2Y(1), receptors in PC12 cells. In Northern blots, only P2Y(2) and P2Y(12) were detectable. Differentiation with NGF did not alter these hybridization signals and left the nucleotide inhibition of adenylyl cyclase unchanged. We conclude that P2Y(12) receptors are expressed in neuronal cells and inhibit adenylyl cyclase activity.

Growth hormone can regulate functions of porcine ovarian granulosa cells through the cAMP/protein kinase A system

The action of growth hormone (GH) on the production of hormones, growth factors, growth factor binding protein and the occurrence of apoptosis in porcine ovarian granulosa cells, as well as the role of cAMP-stimulated protein kinase A (PKA) in the mediation of these effects, were studied. For this purpose, the effects of exogenous pGH (1–10,000 ng/ml), PKA blockers KT5720 (100 ng/ml) and Rp-cAMPS (1 μmol), alone and in combination, on insulin-like growth factor type I (IGF-I), insulin-like binding protein 3 (IGFBP-3), oxytocin (OT) and prostaglandin F alpha (PGF) secretion, PKA and cAMP response element binding transcription factor (CREB) content and the occurrence of apoptosis were investigated. It was found (using RIA/IRMA) that GH addition to culture medium significantly stimulated IGF-I and PGF release and inhibited IGFBP-3 and OT secretion. GH significantly decreased the incidence of apoptosis (TUNEL method) in cultured cells. Immunocytochemical study and Western immunoblotting showed, that addition of GH caused a dramatic increase in the accumulation of immunoreactive PKA within the cells, whilst Western blotting did not reveal marked influence of GH on content of CREB in cell lysates. PKA blockers, given alone, were able to decrease IGFBP-3 output (Rp-cAMPS, but not KT5720), reduce basal OT release (either Rp-cAMPS and KT5720) and increase PGF accumulation (KT5720, but not Rp-cAMPS). Furthermore, PKA blockers were able to prevent stimulatory effects of GH on IGF-I and PGF release, and inhibitory effect of GH on IGFBP-3, OT output and on apoptosis. These observations suggest the involvement of GH and a PKA-dependent intracellular mechanism in the control of IGF-I, IGFBP-3, OT, PGF, cAMP and apoptosis in porcine ovarian granulosa cells. Stimulation of PKA by GH and the prevention of GH-induced effects by PKA blockers suggest that both stimulatory and inhibitory effects of GH on porcine ovarian cells are probably mediated by the cAMP/PKA system.

Changes of inducible nitric oxide synthase in aortic cells during the development of hypertension: Effect of angiotensin II

Nitric oxide (NO) generation by inducible nitric oxide synthase (iNOS) in the vascular smooth muscle cells (VSMC), may play a role in blood vessel tone regulation. Lipopolysaccharide (LPS) induced iNOS activity and subsequent nitrite production by cultured aortic VSMC, from SHR with an established chronic blood pressure elevation (adult SHR) or during the period preceding the development of hypertension (young SHR) and from age-matched normotensive Wistar (W) rats were compared. Angiotensin II (Ang II) effect was also evaluated. Both basal LPS-induced iNOS activity and nitrite accumulation were significantly lower in young SHR VSMC compared to young W rat cells. In contrast, adult hypertensive and normotensive rat cells did not differ in NO generation. Besides, young SHR cells exhibited a significant smaller iNOS activity and nitrites than adult SHR cells. After 24 h-incubation with Ang II, both variables were markedly reduced in all groups. The proportional reduction of iNOS activity and nitrites by Ang II was not different between hypertensive and normotensive rat cells, at any age. However, this Ang II inhibitory effect was greater in both adult SHR and W cells than in VSMC from young rats. In conclusion, a reduced LPS-induced iNOS activity and NO generation was observed in VSMC form spontaneously hypertensive rats before the raise of blood pressure, but not in adult hypertensive rat cells. Additionally, an inhibitory effect of angiotensin II on these variables is described. We can speculate that the impairment in vascular smooth muscle NO production precedes the development of hypertension in SHR and may play a pathophysiologic role in the early blood pressure elevation in genetically hypertensive rats.