Basal Intracellular cAMP Levels Research Articles

Gs活性化型受容体GPR3がマウス視神経障害後の軸索再生に与える影響

G protein-coupled receptor 3 (GPR3) is highly expressed in various neurons, and unique in its ability to constitutively activate the Gαs protein without the addition of ligands, which elevates the basal level of intracellular cAMP. In our earlier study, we clarified that GPR3 play a role in both neurite outgrowth and neuronal survival in developing cerebellar granular neurons. However, the potential role of GPR3 in the axon regeneration still remains unclear. Herein, we investigated the possible involvement of GPR3 in axonal regeneration after optic nerve injury in mice. GPR3 expression was observed in the retinal ganglion cells (RGCs). GPR3 expression was increasingly expressed during development in the mouse primary retinal ganglion neurons (RGNs). When GPR3 expression was suppressed by siRNA, both neurite outgrowth and survival in RGNs were significantly inhibited. In the pressure-induced retinal ischemia model, the number of RGCs and the inner plexiform layer thickness were significantly reduced in the GPR3 knockout mice compared to those in wild-type mice 7 days after the induction of transient retinal ischemia. In addition, regenerating axon was significantly increased 4 weeks after optic nerve crush (ONC) when GPR3 was overexpressed in RGCs using adeno-associated virus. Furthermore, axonal regeneration was dramatically augmented when Zymosan was inoculated in the GPR3-overexpressed retina. These results suggest that GPR3 play a potential role in accelerating axonal regeneration after ONC in mouse.

Signaling and functions of G-protein-coupled receptor 3 in cerebellar granular neurons

G-protein-coupled receptor 3 (GPR3) is a member of the class A rhodopsin-type GPCR family and is highly expressed in various neurons. A unique feature of GPR3 is its ability to constitutively activate the Gαs protein without the addition of ligands, which results in the elevation of the basal level of intracellular cAMP. During the development of the cerebellum, GPR3 expression is upregulated in cerebellar granular neurons (CGNs) and maintained thereafter. In our previous studies, we showed that the intrinsic expression of GPR3 in CGNs is highly associated with neurite outgrowth, neurite differentiation, and neuronal survival. Recently, we have focused on the possible signaling pathways associated with GPR3-mediated neurite outgrowth in CGNs. Interestingly, GPR3-mediated neurite outgrowth is mediated by not only PKA-dependent signaling pathways but also PI3K-mediated signaling pathways. Moreover, the Gβγ-mediated signaling pathway is involved in GPR3-mediated neurite outgrowth. These results suggested that neural expression of GPR3 stimulates multiple downstream signaling pathways, contributing to the maintenance of homeostasis in neurons. Further precise analyses of constitutively active GPCRs may help in unveiling novel neuronal functions.

The effects and possible mechanism of β2AR gene expression in cardiocytes of canines with heart failure.

The objective of the present study was to observe the changes of β2-adrenergic receptor (β2AR) protein expression in a canine model of heart failure (HF), and the function of cardiocytes after transfection with Adv-β2AR. The canine model of chronic HF was induced by rapid right ventricular pacing and cardiocytes were isolated with collagenase II. Cardiocytes were transfected with Adv-β2AR to observe contractile function with a motion edge-detection system of single cells. Expression of β2AR protein in cardiocytes was measured by immunoblotting and the levels of intracellular cAMP were measured by ELISA. Compared with the control group (the sham group), the expression of β2AR protein in HF cardiocytes did not change, but the basal (1 mM Ca2+) contraction amplitude percentage (1.809±0.922 vs. 1.120±0.432%, P<0.05), the maximum contraction amplitude percentage (14.855±2.377 vs. 10.784±2.675%, P<0.01) and the basal levels of intracellular cAMP (9.39±2.54 vs. 5.26±0.95 pmol/ml, n=6, P<0.05) of HF cardiocytes were significantly decreased. However, when HF cardiocytes were transfected with Adv-β2AR and cultured for 48 h, compared with the non-transfected group, the basal contraction amplitude percentage (0.851±0.324 vs. 1.629±0.522%, P<0.05), the maximum contraction amplitude percentage (9.260±2.208% vs. 12.205±1.437%, P<0.01) and the basal levels of intracellular cAMP (5.26±0.95 vs. 9.03±1.03 pmol/ml, n=6, P<0.05) of cardiocytes in the transfected group were significantly increased. In conclusion, the expression of β2AR protein in HF cardiocytes did not change, but contraction function was impaired. The moderate overexpression of β2AR gene in the HF cardiocytes increased the levels of intracellular cAMP and improved contraction function.

Selective Estrogen Receptor Modulators: Cannabinoid Receptor Inverse Agonists with Differential CB1 and CB2 Selectivity.

Selective estrogen receptor modulators (SERMs) are used to treat estrogen receptor (ER)-positive breast cancer and osteoporosis. Interestingly, tamoxifen and newer classes of SERMs also exhibit cytotoxic effects in cancers devoid of ERs, indicating a non-estrogenic mechanism of action. Indicative of a potential ER-independent target, reports demonstrate that tamoxifen binds to cannabinoid receptors (CBRs) with affinity in the low μM range and acts as an inverse agonist. To identify cannabinoids with improved pharmacological properties relative to tamoxifen, and further investigate the use of different SERM scaffolds for future cannabinoid drug development, this study characterized the affinity and activity of SERMs in newer structural classes at CBRs. Fourteen SERMs from five structurally distinct classes were screened for binding to human CBRs. Compounds from four of five SERM classes examined bound to CBRs. Subsequent studies fully characterized CBR affinity and activity of one compound from each class. Ospemifine (a triphenylethylene) selectively bound to CB1Rs, while bazedoxifine (an indole) bound to CB2Rs with highest affinity. Nafoxidine (a tetrahydronaphthalene) and raloxifene (RAL; a benzothiaphene) bound to CB1 and CB2Rs non-selectively. All four compounds acted as inverse agonists at CB1 and CB2Rs, reducing basal G-protein activity with IC50 values in the nM to low μM range. Ospemifine, bazedoxifene and RAL also acted as inverse agonists to elevate basal intracellular cAMP levels in intact CHO-hCB2 cells. The four SERMs examined also acted as CB1 and CB2R antagonists in the cAMP assay, producing rightward shifts in the concentration-effect curve of the CBR agonist CP-55,940. In conclusion, newer classes of SERMs exhibit improved pharmacological characteristics (e.g., in CBR affinity and selectivity) relative to initial studies with tamoxifen, and thus suggest that different SERM scaffolds may be useful for development of safe and selective drugs acting via CBRs.

Sex differences in SR Ca2 + release in murine ventricular myocytes are regulated by the cAMP/PKA pathway

Previous studies have shown that ventricular myocytes from female rats have smaller contractions and Ca2+ transients than males. As cardiac contraction is regulated by the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway, we hypothesized that sex differences in cAMP contribute to differences in Ca2+ handling. Ca2+ transients (fura-2) and ionic currents were measured simultaneously (37°C, 2Hz) in ventricular myocytes from adult male and female C57BL/6 mice. Under basal conditions, diastolic Ca2+, sarcoplasmic reticulum (SR) Ca2+ stores, and L-type Ca2+ current did not differ between the sexes. However, female myocytes had smaller Ca2+ transients (26% smaller), Ca2+ sparks (6% smaller), and excitation–contraction coupling gain in comparison to males (23% smaller). Interestingly, basal levels of intracellular cAMP were lower in female myocytes (0.7±0.1 vs. 1.7±0.2fmol/μg protein; p<0.001). Importantly, PKA inhibition (2μM H-89) eliminated male–female differences in Ca2+ transients and gain, as well as Ca2+ spark amplitude. Western blots showed that PKA inhibition also reduced the ratio of phospho:total RyR2 in male hearts, but not in female hearts. Stimulation of cAMP production with 10μM forskolin abolished sex differences in cAMP levels, as well as differences in Ca2+ transients, sparks, and gain. To determine if the breakdown of cAMP differed between the sexes, phosphodiesterase (PDE) mRNA levels were measured. PDE3 expression was similar in males and females, but PDE4B expression was higher in female ventricles. The inhibition of cAMP breakdown by PDE4 (10μM rolipram) abolished differences in Ca2+ transients and gain. These findings suggest that female myocytes have lower levels of basal cAMP due, in part, to higher expression of PDE4B. Lower cAMP levels in females may attenuate PKA phosphorylation of Ca2+ handling proteins in females, and may limit positive inotropic responses to stimulation of the cAMP/PKA pathway in female hearts.

The Diadenosine Homodinucleotide P18 Improves In Vitro Myelination in Experimental Charcot‐Marie‐Tooth Type 1A

Charcot-Marie-Tooth 1A (CMT1A) is a demyelinating hereditary neuropathy whose pathogenetic mechanisms are still poorly defined and an etiologic treatment is not yet available. An abnormally high intracellular Ca(2+) concentration ([Ca(2+)]i) occurs in Schwann cells from CMT1A rats (CMT1A SC) and is caused by overexpression of the purinoceptor P2X7. Normalization of the Ca(2+) levels through down-regulation of P2X7 appears to restore the normal phenotype of CMT1A SC in vitro. We recently demonstrated that the diadenosine 5',5'''-P1, P2-diphosphate (Ap2A) isomer P18 behaves as an antagonist of the P2X7 purinergic receptor, effectively blocking channel opening induced by ATP. In addition, P18 behaves as a P2Y11 agonist, inducing cAMP overproduction in P2Y11-overexpressing cells. Here we investigated the in vitro effects of P18 on CMT1A SC. We observed that basal levels of intracellular cAMP ([cAMP]i), a known regulator of SC differentiation and myelination, are significantly lower in CMT1A SC than in wild-type (wt) cells. P18 increased [cAMP]i in both CMT1A and wt SC, and this effects was blunted by NF157, a specific P2Y11 antagonist. Prolonged treatment of organotypic dorsal root ganglia (DRG) cultures with P18 significantly increased expression of myelin protein zero, a marker of myelin production, in both CMT1A and wt cultures. Interestingly, P18 decreased the content of non-phosphorylated neurofilaments, a marker of axonal damage, only in CMT1A DRG cultures. These results suggest that P2X7 antagonists, in combination with [cAMP]i-increasing agents, could represent a therapeutic strategy aimed at correcting the molecular derangements causing the CMT1A phenotype.

Inverse Agonist and Pharmacochaperone Properties of MK-0524 on the Prostanoid DP1 Receptor

Prostaglandin D2 (PGD2) acts through two G protein-coupled receptors (GPCRs), the prostanoid DP receptor and CRTH2 also known as DP1 and DP2, respectively. Several previously characterized GPCR antagonists are now classified as inverse agonists and a number of GPCR ligands are known to display pharmacochaperone activity towards a given receptor. Here, we demonstrate that a DP1 specific antagonist, MK-0524 (also known as laropiprant), decreased basal levels of intracellular cAMP produced by DP1, a Gαs-coupled receptor, in HEK293 cells. This reduction in cAMP levels was not altered by pertussis toxin treatment, indicating that MK-0524 did not induce coupling of DP1 to Gαi/o proteins and that this ligand is a DP1 inverse agonist. Basal ERK1/2 activation by DP1 was not modulated by MK-0524. Interestingly, treatment of HEK293 cells expressing Flag-tagged DP1 with MK-0524 promoted DP1 cell surface expression time-dependently to reach a maximum increase of 50% compared to control after 24 h. In contrast, PGD2 induced the internalization of 75% of cell surface DP1 after the same time of stimulation. The increase in DP1 cell surface targeting by MK-0524 was inhibited by Brefeldin A, an inhibitor of transport from the endoplasmic reticulum-Golgi to the plasma membrane. Confocal microscopy confirmed that a large population of DP1 remained trapped intracellularly and co-localized with calnexin, an endoplasmic reticulum marker. Redistribution of DP1 from intracellular compartments to the plasma membrane was observed following treatment with MK-0524 for 24 h. Furthermore, MK-0524 promoted the interaction between DP1 and the ANKRD13C protein, which we showed previously to display chaperone-like effects towards the receptor. We thus report that MK-0524 is an inverse agonist and a pharmacochaperone of DP1. Our findings may have important implications during therapeutic treatments with MK-0524 and for the development of new molecules targeting DP1.

Roflumilast-N-oxide Induces Surfactant Protein Expression in Human Alveolar Epithelial Cells Type II

Surfactant proteins (SPs) are important lipoprotein complex components, expressed in alveolar epithelial cells type II (AEC-II), and playing an essential role in maintenance of alveolar integrity and host defence. Because expressions of SPs are regulated by cyclic adenosine monophosphate (cAMP), we hypothesized that phosphodiesterase (PDE) inhibitors, influence SP expression and release. Analysis of PDE activity of our AEC-II preparations revealed that PDE4 is the major cAMP hydrolysing PDE in human adult AEC-II. Thus, freshly isolated human AEC-II were stimulated with two different concentrations of the PDE4 inhibitor roflumilast-N-oxide (3 nM and 1 µM) to investigate the effect on SP expression. SP mRNA levels disclosed a large inter-individual variation. Therefore, the experiments were grouped by the basal SP expression in low and high expressing donors. AEC-II stimulated with Roflumilast-N-oxide showed a minor increase in SP-A1, SP-C and SP-D mRNA mainly in low expressing preparations. To overcome the effects of different basal levels of intracellular cAMP, cyclooxygenase was blocked by indomethacin and cAMP production was reconstituted by prostaglandin E2 (PGE2). Under these conditions SP-A1, SP-A2, SP-B and SP-D are increased by roflumilast-N-oxide in low expressing preparations. Roflumilast-N-oxide fosters the expression of SPs in human AEC-II via increase of intracellular cAMP levels potentially contributing to improved alveolar host defence and enhanced resolution of inflammation.

GPR3 Receptor, a Novel Actor in the Emotional-Like Responses

GPR3 is an orphan G protein-coupled receptor endowed with constitutive Gs signaling activity, which is expressed broadly in the central nervous system, with maximal expression in the habenula. We investigated the consequences of its genetic deletion in several behavioral paradigms and on neurotransmission. Compared to wild-type, hippocampal neurons from Gpr3−/− mice displayed lower basal intracellular cAMP levels, consistent with the strong constitutive activity of GPR3 in transiently transfected cells. Behavioral analyses revealed that Gpr3−/− mice exhibited a high level of avoidance of novel and unfamiliar environment, associated with increased stress reactivity in behavioral despair paradigms and aggressive behavior in the resident-intruder test. On the contrary, no deficit was found in the learning ability to avoid an aversive event in active avoidance task. The reduced ability of Gpr3 −/− mice to cope with stress was unrelated to dysfunction of the hypothalamic-pituitary-adrenal axis, with Gpr3−/− mice showing normal corticosterone production under basal or stressful conditions. In contrast, dramatic alterations of monoamine contents were found in hippocampus, hypothalamus and frontal cortex of Gpr3−/− mice. Our results establish a link between tonic stimulation of the cAMP signaling pathway by GPR3 and control of neurotransmission by monoamines throughout the forebrain. GPR3 qualifies as a new player in the modulation of behavioral responses to stress and constitutes a novel promising pharmacological target for treatment of emotional disorders.

Weak evidence of thyrotropin receptors in primary cultures of human osteoblast-like cells.

Extra-thyroidal thyrotropin (TSH) receptors (TSHRs) have been demonstrated in several tissues and cells, including human and rat osteosarcoma cell lines. We have explored whether human TSHR (hTSHRs) also are present in primary cultures of human osteoblast-like (hOB) cells. [(125) I]TSH binding was limited in hOB cells, but somewhat higher in UMR 106-01 cells and considerably higher in hTSHR-transfected CHO cells. In hOB cells, the basal intracellular cAMP levels increased 282% after stimulation with 10 U/L TSH. In the hTSHR-transfected CHO cells, the cAMP increase was 3030% in response to 10 U/L TSH and 1240% after 1 U/L TSH. Free cytoplasmic calcium did not change in response to TSH in hOB cells. HTSHR mRNA was detected in hOB cells from 3/4 bone by reverse transcriptase polymerase chain reaction RT-PCR and nucleotide sequencing HTSHR mRNA, but could not be demonstrated with the RNase protection technique in hOB cells from 5 different donors. In conclusion, even after the use of several methods, we have found only weak evidence for expression and presence of functionally active hTSHR in hOB cells. Given the low level of expression, specific binding and cAMP signaling, we suggest that it is unlikely that circulating TSH plays a physiological role for bone metabolism mediated through osteoblasts.

Dopamine modulates the plasticity of mechanosensory responses in Caenorhabditis elegans.

Dopamine-modulated behaviors, including information processing and reward, are subject to behavioral plasticity. Disruption of these behaviors is thought to support drug addictions and psychoses. The plasticity of dopamine-mediated behaviors, for example, habituation and sensitization, are not well understood at the molecular level. We show that in the nematode Caenorhabditis elegans, a D1-like dopamine receptor gene (dop-1) modulates the plasticity of mechanosensory behaviors in which dopamine had not been implicated previously. A mutant of dop-1 displayed faster habituation to nonlocalized mechanical stimulation. This phenotype was rescued by the introduction of a wild-type copy of the gene. The dop-1 gene is expressed in mechanosensory neurons, particularly the ALM and PLM neurons. Selective expression of the dop-1 gene in mechanosensory neurons using the mec-7 promoter rescues the mechanosensory deficit in dop-1 mutant animals. The tyrosine hydroxylase-deficient C. elegans mutant (cat-2) also displays these specific behavioral deficits. These observations provide genetic evidence that dopamine signaling modulates behavioral plasticity in C. elegans.

Rapid induction of cAMP/PKA pathway during retinoic acid-induced acute promyelocytic leukemia cell differentiation.

The second messenger cyclic adenosine monophosphate (cAMP) plays an important role in cell proliferation, differentiation and apoptosis. In the present work, we evaluated the cAMP signaling in acute promyelocytic leukemia (APL) cells in the context of differentiation induced by all-trans retinoic acid (ATRA). There was a marked increase in the intracellular cAMP level within a few minutes after treatment with ATRA in APL cell line NB4 and fresh APL cells, whereas no such phenomenon was observed in NB4-R1 cells that are resistant to ATRA-induced maturation. In addition, the basal level of intracellular cAMP was lower in NB4-R1 than in NB4 cells. Mechanistic study showed that this induction of cAMP was mediated through the activation of adenylate cyclase. Moreover, we found that cAMP-dependent protein kinase (PKA) activity was quickly upregulated in parallel in ATRA-treated NB4 cells, and the phosphorylation of RARalpha by PKA could increase its transactivation effect. Use of H-89, an inhibitor of PKA, could partially suppress the transcriptional expression of ATRA target genes and ATRA-induced differentiation of APL cells. Taken together, we suggested a crosstalk between ATRA-induced cytosolic pathway and nuclear pathway in APL cell differentiation.

Endothelial Nitric-oxide Synthase Enhances Lipopolysaccharide-stimulated Tumor Necrosis Factor-α Expression via cAMP-mediated p38 MAPK Pathway in Cardiomyocytes

The purpose of this study was to investigate the role of endothelial nitric-oxide synthase (eNOS), cAMP, and p38 MAPK in tumor necrosis factor-alpha (TNF-alpha) expression induced by lipopolysaccharide (LPS). LPS dose- and time-dependently induced phosphorylation of p38 MAPK and TNF-alpha expression in neonatal mouse cardiomyocytes. TNF-alpha expression was preceded by p38 MAPK phosphorylation, and selective inhibition of p38 MAPK abrogated LPS-induced TNF-alpha expression. Deficiency in eNOS decreased basal and LPS-stimulated TNF-alpha expression in cardiomyocytes. NOS inhibitor l-NAME attenuated LPS-induced p38 MAPK phosphorylation and TNF-alpha production in wild-type cardiomyocytes, whereas NO donor 2,2'-(hydroxynitrosohydrazono)bis-ethanamine (DETA-NO) (2 microm) or overexpression of eNOS by adenoviral gene transfer restored the response of eNOS(-/-) cardiomyocytes to LPS. These effects of NO were mediated through cAMP-dependent pathway based on the following facts. First, deficiency in eNOS decreased basal levels of intracellular cAMP, and DETA-NO elevated intracellular cAMP levels in eNOS(-/-) cardiomyocytes. Second, a cAMP analogue 8-Br-cAMP mimicked the effect of NO in eNOS(-/-) cardiomyocytes. Third, either inhibition of cAMP or cAMP-dependent protein kinase attenuated LPS-stimulated p38 MAPK phosphorylation and TNF-alpha production in wild-type cardiomyocytes. In conclusion, eNOS enhances LPS-stimulated TNF-alpha expression in cardiomyocytes. Activation of p38 MAPK is essential in LPS-stimulated TNF-alpha expression. Moreover, the effects of NO on LPS-stimulated TNF-alpha expression are mediated through cAMP/cAMP-dependent protein kinase-dependent p38 MAPK pathway in neonatal cardiomyocytes.

Cloning and characterization of a novel human 5-HT 4 receptor variant that lacks the alternatively spliced carboxy terminal exon. RT-PCR distribution in human brain and periphery of multiple 5-HT 4 receptor variants

We have cloned a novel C-terminal splice variant of serotonin 5-HT 4 receptors from human hippocampus. The deduced protein extends only one aminoacid past the splicing point. We propose to call the novel variant h5-HT 4(n) since it contains none of the C-terminal exons alternatively spliced in other variants. The pharmacological profile of h5-HT 4(n) stably expressed in HeLa cells is in agreement with other reported variants. Stably transfected cells showed increased basal levels of intracellular cAMP in absence of agonist, indicating constitutive activity of the expressed receptors. 5-HT induced robust increases of intracellular cAMP. The 5-HT 4 receptor antagonist GR 113808 blocked the effects of 5-HT and brought intracellular cAMP below basal constitutive levels, indicating inverse agonism of this compound in this system. The RT-PCR distribution of all known human C-terminal splice variants in human brain regions and periphery showed complex patterns of variant expression, with the novel variant h5-HT 4(n) being widely and abundantly expressed.

Stimulation of mitogen-activated protein kinase by gonadotropin-releasing hormone in human granulosa-luteal cells.

The present study investigated the activation of mitogen-activated protein kinases (MAPKs) by a GnRH agonist (GnRHa) in human granulosa-luteal cells (hGLCs). The phosphorylation state of p44 and p42 MAPK was examined using antibodies that distinguish phospho-p44/42 MAPK (Thr(202)/Tyr(204)) from total p44/42 MAPK (activated plus inactivated). Activation of MAPK by GnRHa was observed within 5 min and was sustained for 60 min after treatment. GnRHa stimulated MAPK activation in a dose-dependent manner, with maximum stimulation (6.7-fold over basal levels) at 10(-7) M. Pretreatment with a protein kinase C (PKC) inhibitor, GF109203X, completely blocked GnRHa-induced MAPK activation. In addition, pretreatment with a PKC activator, phorbol-12-myristate 13-acetate, potentiated GnRH-induced MAPK activation. These results indicate that GnRHa stimulates MAPK activation through a PKC-dependent pathway in hGLCs, possibly coupled to G(q)alpha protein. MAPK activation was also observed in response to 8-bromo-cAMP or cholera toxin, but not pertussis toxin. Forskolin (50 microM) substantially stimulated a rapid cAMP accumulation, whereas GnRHa (10(-7) M) or pertussis toxin (100 mg/ml) did not affect basal intracellular cAMP levels. Cotreatment of GnRHa (10(-7) M) did not attenuate forskolin- or hCG-stimulated cAMP accumulation. These results suggest that the GnRH receptor is probably not coupled to G(s)alpha or G(i)alpha in hGLCs. Finally, GnRHa (10(-7) M) stimulated a significant increase in Elk-1 phosphorylation and c-fos messenger RNA expression, as revealed by an in vitro kinase assay and Northern blot analysis, respectively. These results clearly demonstrate that GnRH activates the MAPK cascade through a PKC-dependent pathway in the human ovary.

Visualizing differences in ligand regulation of wild-type and constitutively active mutant beta(2)-adrenoceptor-green fluorescent protein fusion proteins.

Fusion proteins were generated by attachment of green fluorescent protein (GFP) to the C-terminal tail of either the wild-type human beta(2)-adrenoceptor or a form with enhanced constitutive activity. Sustained treatment of HEK293 cells stably expressing the constitutively active mutant (CAM) beta(2)-adrenoceptor-GFP with the inverse agonist betaxolol resulted in a marked up-regulation of the fusion protein that could be monitored by both fluorescence and immunoblotting of membrane fractions. This was not observed for the wild-type beta(2)-adrenoceptor-GFP. Addition of the agonist isoprenaline to CAM beta(2)-adrenoceptor-GFP expressing cells previously treated with betaxolol resulted in rapid internalization of the receptor into punctate intracellular vesicles in a manner similar to wild-type beta(2)-adrenoceptor-GFP. A range of "beta-blockers" replicated the up-regulation of the CAM beta(2)-adrenoceptor-GFP, although pharmacological specificity was maintained, as it was not produced by alpha(1)- and alpha(2)-adrenoceptor-selective antagonists/inverse agonists. Parallel intact cell binding studies with [(3)H]dihydroalprenolol confirmed up-regulation of the CAM beta(2)-adrenoceptor-GFP by betaxolol but failed to predict the optically monitored up-regulation produced by high concentrations of alprenolol. The cellular distribution of the up-regulated CAM beta(2)-adrenoceptor-GFP was not identical after sustained treatment of the cells with different beta-blockers. Inverse agonists, able to reduce basal intracellular cAMP levels, such as betaxolol and ICI118551, resulted in both increased plasma membrane receptor and increased diffuse intracellular staining. In contrast, treatment with labetolol and alprenolol resulted in a significant fraction of the intracellular receptor displaying a punctate distribution pattern. These ligands displayed substantial agonism to stimulate intracellular cAMP levels via the CAM beta(2)-adrenoceptor-GFP.

An autocrine or a paracrine role of adrenomedullin in modulating cardiac fibroblast growth.

The aim of the present study was to determine the role of adrenomedullin (AM) in cardiac fibroblasts. The production and secretion of AM were examined in cultured neonatal rat cardiac fibroblasts, and the effects of AM on proliferation and protein synthesis of these cells were assessed by [3H]thymidine and [3H]phenylalanine incorporation, respectively. Cultured cardiac fibroblasts secreted AM into the medium time-dependently at a rate of 20.3 +/- 3.0 fmol/5 x 10(4) cells/48 h, mean +/- S.D. Northern blot analysis showed expression of preproAM mRNA of 1.6 kb in these cells. In addition, 10(-6) mol/l of angiotensin II (Ang II) and endothelin-1 (ET-1) significantly increased the AM secretion by 55 and 48%, respectively. Synthetic AM significantly reduced 10(-6) mol/l Ang II- or 10(-7) mol/l ET-1-stimulated [3H]thymidine and [3H]phenylalanine incorporation in a dose-dependent manner, and these effects were attenuated by a calcitonin gene-related peptide (CGRP) type 1 receptor antagonist, CGRP(8-37). Synthetic AM also had a dose-dependent stimulatory effect on cAMP accumulation in these cells, which was significantly attenuated by CGRP(8-37). A cAMP analogue, 8-bromo-cAMP, mimicked the AM effects, inhibiting the Ang II-stimulated [3H]thymidine and [3H]phenylalanine incorporation. Blockage of the effect of endogenous AM by anti-AM monoclonal antibody not only significantly reduced the basal level of intracellular cAMP, but also enhanced the [3H]thymidine and [3H]phenylalanine incorporation into the cells. Cultured neonatal rat cardiac fibroblasts produce and secrete AM, and the secreted AM may inhibit proliferation and protein synthesis of these cells. AM may exert these inhibitory effects partly by elevating intracellular cAMP. It is suggested that AM has an important role in modulating the growth of cardiac fibroblasts in an autocrine or a paracrine manner.

A Novel Luteinizing Hormone Receptor Mutation in a Patient with Familial Male-Limited Precocious Puberty: Effect of the Size of a Critical Amino Acid on Receptor Activity

Familial male-limited precocious puberty (FMPP) is a form of luteinizing hormone-releasing hormone (LHRH)-independent isosexual precocious puberty caused by gain-of-function mutations of the luteinizing hormone/chorionic gonadotropin receptor (hLHR). The most common mutation is 1733 A>G, which causes substitution of Asp-578 by Gly. In this study, a male infant presented at the age of 20 months with accelerated sexual development was analyzed for the presence of activating mutations of the hLHR. Analysis of exon 11 of the hLHR gene by genomic polymerase chain reaction (PCR), asymmetric PCR, and dideoxy sequencing identified a single base substitution, 1734 T>A, which led to the replacement of Asp-578 by Glu. The same mutation was found in the mother. Expression of the mutated hLHR in HEK 293 cells demonstrated elevated basal levels of intracellular cAMP in the transfected cells confirming the constitutive activating nature of the mutated hLHR. A possible genotype-phenotype relationship of the hLHR mutations was examined by a comparison of thein vitroactivities of the hLHRs carrying the Asp578Gly, Asp578Tyr, Asp578Trp, and Asp578Glu mutations in HEK 293 cells. A positive correlation between the size of the substituting amino acid and the basal level of intracellular cAMP of cells expressing the mutated receptor was demonstrated.

Adrenomedullin stimulates cyclic AMP production in the airway epithelial cells of guinea-pigs and in the human epithelial cell line

This study was designed to examine the effects of adrenomedullin (AM) on airway epithelial cells. Primary cultures of guinea-pig tracheal epithelial cells and the human bronchiolar epithelial cell line NCI-H441 were used. Intracellular cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), prostaglandin E2 (PGE2), and stable end-products of nitric oxide were assayed. Adrenomedullin (10− 6 mol/L) stimulated cAMP production in guinea-pig epithelial cells. Indomethacin (10− 5 mol/L) significantly decreased the basal level of intracellular cAMP in guinea-pig epithelial cells, but not in NCI-H441 cells. However, AM did not stimulate production of PGE2, a major product that can increase cAMP formation. In the case of NCI-H441 cells, AM (10− 8 – 10− 6 mol/L) did not significantly affect intracellular cGMP levels or nitrite content in conditioned medium. Adrenomedullin and calcitonin gene-related peptide (CGRP) each stimulated cAMP production in NCI-H441 cells, but AM-stimulated cAMP production was antagonized by the CGRP fragment CGRP8–37. These findings suggest that AM stimulates cAMP production and functionally competes with CGRP for binding sites in airway epithelial cells, at least in human epithelial cells, but that it does not stimulate the release of PGE2 and nitric oxide. Though cyclooxygenase products contribute to some extent to cAMP formation in guinea-pigs, AM independently stimulates intracellular cAMP formation in airway epithelial cells.

Adenosine modulates cell proliferation in human colonic adenocarcinoma. I. Possible involvement of adenosine A 1 receptor subtypes in HT29 cells

Several lines of evidence suggest that extracellular adenosine interacting with specific cell surface receptors may influence cell growth and differentiation of cancer cells in culture. The data presented here demonstrate that various treatments of human colonic adenocarcinoma HT29 cells in the presence of exogenously added adenosine deaminase, which converts extracellular adenosine into inosine, resulted in a significant decrease of the proliferation. Cell growth inhibition was also observed in the presence of adenosine A 1 receptor antagonists. These various treatments also induced a significant elevation of basal intracellular cAMP levels. This strongly indicated that extracellular adenosine was maintaining low intracellular cAMP levels in HT29 cells. A partial pharmacological characterization of the binding of the adenosine A 1 receptor agonist [ 3 H ]CCPA (2-chloro- N 6-cyclopentyl[2,3,4,5- 3 H ]adenosine), and the adenosine A 1 receptor antagonist [ 3 H ]DPCPX (cyclopentyl-1,3-dipropyl[2,3- 3 H ]xanthine), to HT29 cells is also provided. Together the data support the idea that A 1-adenosine receptors are expressed in HT29 cells and might mediate part of the above described effects of adenosine on cell proliferation.