Adenosine Monophosphate Response Research Articles

Follicle stimulating hormone promotes production of renin through its receptor in juxtaglomerular cells of kidney

BackgroundPost-menopausal hypertension has been attributed solely to declining estrogen levels. The purpose of the research is to elucidate the mechanism by which follicle stimulating hormone(FSH) increases renin production involved in the regulation of blood pressure.MethodsThe expression of follicle stimulating hormone receptors (FSHRs) in renal juxtaglomerular cells and a As4.1 juxtaglomerular mouse cell line was evaluated. We established a mouse model by ovariectomy (OVX). Ovariectomized mice were treated with gonadotropin-releasing hormone agonist (GnRHa) (OVX + GnRHa). Ovariectomized mice initially received physiological doses of estrogen and were then injected with recombinant FSH (OVX + E + FSH).ResultsWe found that FSHR was expressed in mouse renal juxtaglomerular cells labeled by renin antibody and in As4.1 cells. FSH promoted renin synthesis via Gsα-coupled FSHRs that activated protein kinase A, cyclic adenosine monophosphate(cAMP) response element-binding protein, extracellular signal-regulated kinase (Erk1/2), Protein kinase B(AKT), and c-Jun N-terminal kinase signaling pathways in As4.1 cells. We found increased serum FSH levels in the ovariectomized mouse with concurrent increases in renin, angiotensin II, heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial blood pressure (MAP). Additionally, increases in serum renin, angiotensin II, HR, SBP, DBP, and MAP were reduced by the additional injection of GnRHa. Exogenous FSH administration completely reversed decreases in renin, angiotensin II, HR, SBP, DBP, and MAP even in mice that received physiological doses of estrogen to maintain normal estradiol levels.ConclusionsElevated FSH stimulates renin production involving a mechanism that may be relevant to the expression of FSH receptors in renal juxtaglomerular cells.

Metabotropic glutamate receptor 5 regulates synaptic plasticity in a chronic migraine rat model through the PKC/NR2B signal

BackgroundThe mechanism of chronic migraine (CM) is complex, central sensitization is considered as one of the pathological mechanism. Synaptic plasticity is the basis of central sensitization. Metabotropic glutamate receptor 5 (mGluR5) plays a vital role in the synaptic plasticity of the central nervous system. However, whether mGluR5 can promote the central sensitization by regulating synaptic plasticity in CM is unknown.MethodsMale Wistar rats were used to establish a CM rat model, and the expression of mGluR5 mRNA and protein were detected by qRT-PCR and western blot. The allodynia was assessed by mechanical and thermal thresholds, and central sensitization was assessed by expression of the phosphorylation of cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) at Serine 133(pCREB-S133) and c-Fos. The synaptic-associated protein postsynaptic density protein 95 (PSD), synaptophysin (Syp), and synaptophysin-1(Syt-1), synaptic ultrastructure, and dendritic spines were detected to explore synaptic plasticity. The expression of PKC, total NR2B(tNR2B), and phosphorylation of NR2B at Tyr1472(pNR2B-Y1472) were detected by western blot.ResultsWe found that the expression of mGluR5 was upregulated in CM rats. Downregulated the mGluR5 with MPEP alleviated the allodynia and reduced the expression of CGRP, pCREB-S133, c-Fos, PSD, Syp and Syt-1 and synaptic transmission. Moreover, the administration of MPEP inhibited the upregulation of PKC and pNR2B-Y1472.ConclusionsThese results indicate that mGluR5 contributes to central sensitization by regulating synaptic plasticity in CM through the PKC/NR2B signal, which suggests that mGluR5 may be a potential therapeutic candidate for CM.

Effects of 5-hydroxymethyl furfural extracted from Rehmannia glutinosa Libosch on the expression of signaling molecules relevant to learning and memory among hippocampal neurons exposed to high concentration of corticosterone

To determine the effects of 5-hydroxymethyl furfural (5-HMF), an extract of Rehmannia glutinosa Libosch, on several down-regulated signaling molecules involved in learning and memory in hippocampal neurons. After cultured for 7 days, primary hippocampal neurons were divided into 5 groups: normal, corticosterone model, RU38486, 5-HMF, and donepezil group. Neuron survival rates were calculated 24 h later using SYTO13-PI double-fluorescence staining and an 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. β-galactosidase activity was also assayed. Protein expressed by the glucocorticoid receptor (GCR), brainderived neurotrophic factor (BDNF), and N-methyl-D-aspartate receptor 2B (NR2B), as well as phosphorylationcyclic adenosine monophosphate (cAMP) response element binding protein (p-CREB), phosphorylation-extracellular signal-regulated kinase (p-ERK), and phosphorylation-synapsin (p-synapsin) were quantified with Western blot. Hippocampal neuron survival rates and the above-mentioned proteins were dramatically decreased (P<0.05), β-galactosidase activity was significantly increased in the model group. but the effect was reversed by 5-HMF, RU38486, and to a lesser extent by donepezil (P<0.05). 5-HMF extracts from the Chinese herb Rehmannia glutinosa Libosch could protect hippocampal neurons from glucocorticoid injury and from down-regulated signaling molecules in the GCR-BDNF-NR2B-p-ERK-p-CREB-p-synapsin signal transduction pathway.

The peripheral cannabinoid receptor 1 antagonist VD60 efficiently inhibits carbon tetrachloride-intoxicated hepatic fibrosis progression

This study investigated a peripheral selective CB₁ antagonist 3,4,22-3-demethoxycarbonyl-3-hydroxylmethyl-4-deacetyl-vindoline 3,4-thionocarbonate (VD60) that efficiently inhibited hepatic fibrosis with lower psychological side effects. A competitive radiolabeled ligand binding experiment and 3'-5'-cyclic adenosine monophosphate (cAMP) response element-driven luciferase analysis were performed to evaluate the antagonistic activity of VD60. Cell viability and collagen production were examined in the human hepatic stellate cell (HSC) line LX-2 and primary cultured rat HSCs. The antifibrotic effects of VD60 were investigated in a CCl₄-induced liver fibrosis mouse model. The concentration of VD60 in the blood and the brain was determined by high-performance liquid chromatography-mass spectrum analysis. Furthermore, the potential underlying mechanisms of VD60 were investigated by Western blot. VD60 selectively competed with the radiolabeled CB1 agonist to bind to CB1. VD60 antagonized CB1 agonist-induced Akt phosphorylation and increased the accumulation of intracellular cAMP. VD60 strongly reduced the expression of α₂(I) pro-collagen mRNA and exerted potent antiproliferative effects on primary HSCs and LX-2 cells. The inhibition of reactive oxygen species production and phosphorylation of Akt, extracellular-signal-regulated kinase (ERK), and Smad3 may explain the underlying mechanisms behind the antiproliferative effect of VD60. Moreover, the in vivo antifibrotic activity of VD60 was confirmed in a CCl4-induced liver fibrosis mouse model. Most importantly, the concentration of VD60 in the peripheral blood was much higher than in the brain, suggesting that VD60 could act as a novel peripheral CB1 antagonist to efficiently inhibit hepatic fibrosis and could be used as a lead compound with low brain side effects in peripheral antifibrotic agents.

Preliminary investigation of the influence of CREB1 gene polymorphisms on cognitive dysfunction in Chinese patients with major depression

Research has increasingly focused on the role of the cyclic adenosine monophosphate (cAMP) response element binding (CREB) protein in learning and memory, particularly its role in cognitive disorders and neurodegeneration, such as Huntington's disease, Alzheimer's disease, Rubinstein–Taybi syndrome, and Coffin–Lowry syndrome. The cognitive dysfunction of patients with major depressive disorder (MDD), which is widely recognized, is not completely in accordance with depressive severity, and the dysfunction persists upon clinical remission in some patients. However, few studies have focused on the role of CREB on cognitive function in patients with MDD. This study aimed to investigate the influence of CREB1 polymorphism on cognitive function in patients with MDD. The current study comprised 113 patients with MDD. The severity of depression was measured using the 17-item Hamilton Depression Rating Scale, and cognitive function was assessed using the Stroop Neuropsychological Screening Test, verbal fluency test, and tests of immediate logical memory and visual reproduction. All subjects were genotyped with regard to CREB1 polymorphisms (rs10932201, rs2551645, rs2254137, rs6740584, and rs2551640). For the verbal fluency test, the results showed significant differences for all single-nucleotide polymorphism genotypic groups. For the Stroop color-word task, a significant difference was found only for rs6740584. No significant differences were found for the Stroop color task, the immediate logical memory test or the visual reproduction test. In conclusion, there was an effect of CREB1 polymorphism on selective attention and retrieval of long-term memory, but not on immediate memory.

Herpes simplex virus type 1 infection activates the Epstein-Barr virus replicative cycle via a CREB-dependent mechanism

The reactivation of latent Epstein-Barr virus (EBV) to lytic replication is important in pathogenesis and requires virus-host cellular interactions. However, the mechanism underlying the reactivation of EBV is not yet fully understood. In the present study, herpes simplex virus type 1 (HSV-1) was shown to induce the reactivation of latent EBV by triggering BZLF1 expression. The BZLF1 promoter (Zp) was not activated by HSV-1 essential glycoprotein-induced membrane fusion. Nevertheless, Zp was activated within 6 h post HSV-1 infection in virus entry-dependent and replication-independent manners. Using a panel of Zp deletion mutants, HSV-1 was shown to promote Zp through a cyclic adenosine monophosphate (cAMP) response element (CRE) located in ZII. The phosphorylated cAMP response element-binding (phos-CREB) protein, the cellular transactivator that binds to CRE, also increased after HSV-1 infection. By transient transfection, cAMP-dependent protein kinase A and HSV-1 US3 protein were found to be capable of activating Zp in CREB- and CRE-dependent manners. The relationship between EBV activation and HSV-1 infection revealed a possible common mechanism that stimulated latent EBV into lytic cycles in vivo.

Abstract 1235: The organic cation transporter SLC22A5 gene is regulated by the cooperative activity of the estrogen receptor and other transcription factors in breast cancer

Abstract The solute carrier family 22 member 5 (SLC22A5) gene encodes a polyspecific organic cation transporter that is associated with many diseases. However, the regulation of SLC22A5 and its relationship with breast cancer is not clearly defined. In a previous global gene expression analysis from our group, SLC22A5 was identified as an estrogen-regulated gene. In this study, we investigated the molecular mechanism by which estrogen receptor (ER) and other transcription factors regulate SLC22A5 gene expression in breast cancer. SLC22A5 is highly expressed in ER-positive breast cancer cell lines and ER-positive mammary tumor tissues compared to ER-negative cells and tissues. SLC22A5 expression is also correlated with the levels of ER expression in breast cancer cell lines and in mammary tumor tissue specimens. SLC22A5 mRNA expression is induced in MCF-7 breast cancer cells within half an hour after estrogen treatment and remains elevated for over 24 hours. Actinomycin D, but not cycloheximide, can inhibit this estrogen induction, indicating new protein synthesis is not needed for induction. These results suggest that estrogen directly activates SLC22A5 transcription. We next cloned the 6kb SLC22A5 promoter and luciferase experiments demonstrated that this portion of the SLC22A5 promoter is not responsive to estrogen treatment. However, we discovered that an intronic enhancer located approximately 9kb from the transcriptional start site confers estrogen responsiveness when inserted downstream of the SLC22A5 gene promoter-luciferase reporter construct. Several transcription factors binding sites, such as a cyclic adenosine monophosphate (cAMP) response element (CRE), an estrogen-response element (ERE), an activator protein-1 (AP-1) site and a half ERE, are juxtaposed in the middle of this intronic enhancer. Mutation or deletion of these elements abolishes the estrogen responsiveness. Chromatin-immunoprecipitation (ChIP) assay demonstrates the recruitment of the transcription factors onto the intronic enhancer region in an estrogen-dependent manner. We next investigated the effect of SLC22A5 knockdown on breast cancer cell growth. Results from these studies show that shRNA knockdown of SLC22A5 inhibits the proliferation of MCF-7 cells. This reduced proliferation is accompanied by decreased cyclin D1 levels. These results suggest that SLC22A5 is an estrogen-induced gene that is involved in regulating breast cell growth. Our results also suggest that this estrogen induction is mediated by a complex intronic enhancer. This estrogen-induced gene may serve as a novel breast cancer biomarker that may be targeted for improved treatment and prevention of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1235.

Cyclic AMP response element-binding protein is implicated in IL-6 production from arthritic synovial cells

Overproduction of interleukin (IL)-6 from synovial cells is critically involved in the pathogenesis of rheumatoid arthritis (RA). Cyclic adenosine monophosphate (AMP) response element-binding protein (CREB), a leucine zipper transcription factor, is expressed at a high level in synovial cells of patients with RA. Although CREB transactivates IL-6 expression in vascular smooth muscle cells, the relation between CREB expression and IL-6 production from arthritic synovial cells remains unclear. In this study, to determine whether CREB is implicated in IL-6 production from arthritic synovial cells, a dominant negative molecule of activation transcription factor 1 (ATF-1) was transfected into synovial cells obtained from arthritic joints of env-pX rats. These transgenic rats carrying the env-pX gene of human T-cell leukemia virus type-1 develop destructive arthritis with high titers of serum rheumatoid factor and are thus regarded as a suitable model of RA. The dominant negative ATF-1 (ATF-1DN) constitutes a heterodimer with CREB and inhibits CREB function, as CREB/ATF-1DN heterodimers no longer bind to the target sequence of CREB. We showed that transfection of ATF-1DN significantly reduced IL-6 production from arthritic synovial cells. These findings suggest that CREB is implicated in IL-6 production from synovial cells and plays an important role in RA pathogenesis.

Relationships of methacholine and adenosine monophosphate responsiveness with serum vascular endothelial growth factor in children with asthma

Airway hyperresponsiveness, which is a characteristic feature of asthma, is usually measured by means of bronchial challenge with direct or indirect stimuli. Vascular endothelial growth factor (VEGF) increases vascular permeability and angiogenesis, leads to mucosal edema, narrows the airway diameter, and reduces airway flow. To examine the relationships between serum VEGF level and airway responsiveness to methacholine and adenosine monophosphate (AMP) in children with asthma. Peripheral blood eosinophil counts, serum eosinophil cationic protein (ECP) concentrations, and serum VEGF concentrations were measured in 31 asthmatic children and 26 control subjects. Methacholine and AMP bronchial challenges were performed on children with asthma. Children with asthma had a significantly higher mean (SD) level of VEGF than controls (361.2 [212.0] vs 102.7 [50.0] pg/mL; P < .001). Blood eosinophil counts and serum ECP levels significantly correlated inversely with AMP provocation concentration that caused a decrease in forced expiratory volume in 1 second of 20% (PC20) (r = -0.474, P =.01; r = -0.442, P =.03, respectively), but not with methacholine PC20 (r = -0.228, P = .26; r = -0.338, P =.10, respectively). Serum VEGF levels significantly correlated with airway responsiveness to AMP (r = -0.462; P = .009) but not to methacholine (r = -0.243; P = .19). Serum VEGF levels were increased in children with asthma and were related to airway responsiveness to AMP but not to methacholine. Increased VEGF levels in asthmatic children may result in increased airway responsiveness by mechanisms related to airway inflammation or increased permeability of airway vasculature.

Effects of Phosphodiesterase Inhibitors on Tension Induced by Norepinephrine and Accumulation of Cyclic Nucleotides in Isolated Human Prostatic Tissue

To further evaluate the mechanism of action of phosphodiesterase (PDE) inhibitors on the human prostate, the effects of PDE4 and PDE5 inhibitors on the tension induced by norepinephrine (NE) and on the intracellular levels of cyclic nucleotides in isolated human prostatic tissue were investigated. Using the organ bath technique, the effects of increasing concentrations (1 nM to 10 microM) of the PDE5 inhibitors sildenafil, tadalafil, and vardenafil and the PDE4 inhibitors rolipram and RP 73401 on the tension induced by NE (40 microM) of prostate strip preparations were investigated. The accumulation of cyclic guanosine monophosphate and cyclic adenosine monophosphate in response to drug exposure was determined by radioimmunoassays. The tension induced by NE was dose dependently reversed by the drugs with the following rank order of efficacy: tadalafil greater than RP 73401 greater than rolipram greater than or equal to vardenafil greater than sildenafil. The maximal reversion of tension values ranged from 52.3% (tadalafil) to 17% (sildenafil). Of the PDE inhibitors, only tadalafil induced a 50% reversion of the initial tension. The most prominent enhancement in tissue cyclic adenosine monophosphate was registered in response to RP 73401 (11-fold), and cyclic guanosine monophosphate levels were significantly elevated by tadalafil, vardenafil, and sildenafil (28-fold, 12-fold, and 3-fold, respectively). Our results have demonstrated that drugs interfering with the cyclic nucleotide-mediated pathways can reverse the tension induced by NE in isolated prostatic tissue and elevate cyclic adenosine monophosphate and cyclic guanosine monophosphate. Our findings serve to explain how PDE inhibitors can affect symptoms of lower urinary tract symptoms and benign prostatic hyperplasia.

Differential activation of cAMP response element binding protein in discrete nucleus accumbens subregions during early and late cocaine sensitization.

The present study examined the differential cocaine-induced activation of the cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) throughout discrete zones of analysis of the nucleus accumbens (NAc) in rats. CREB-dependent gene transcription, which may underlie long-lasting drug-induced changes in behavior and the subjective effects of cocaine, varies depending on the stage of drug exposure or withdrawal and the cell population involved. Using immunohistochemistry, the authors analyzed changes in CREB phosphorylation in the NAc after 5 days of cocaine, a short or long drug-free period, and a subsequent challenge injection. The NAc shell was separated into 5 zones of analysis previously defined by neurochemistry and connectivity. Repeated cocaine resulted in CREB phosphorylation in all analyzed subregions of the NAc excluding the most ventrolateral region of the shell 2 weeks after cessation of repeated cocaine, but rats challenged after 2 drug-free days yielded a more localized activation of CREB in the 3 most dorsomedial zones of the shell. The temporal and anatomical determinants of cocaine-induced CREB activity may indicate functional differences among NAc shell subregions and suggest the involvement of CREB in early and late cocaine effects.

Allergen sensitization and bronchial hyper-responsiveness to adenosine monophosphate in asthmatic patients.

Indirect bronchoprovocation using adenosine monophosphate (AMP) is related to atopic phenotype expression. To evaluate the putative relationship between skin prick allergen sensitization and bronchial hyper-responsiveness to AMP in a retrospective cross-sectional database analysis. We retrospectively evaluated two groups of non-smoking asthmatics (forced expiratory volume in 1 s (FEV)1>/=60% predicted) who were reactive (responders) or unreactive (controls) to inhaled AMP. The main outcome measure was the difference in sensitization to individual allergens and the total atopic load according to the presence or absence of bronchial hyper-responsiveness. We initially identified 180 (44%) non-smoking asthmatics with PC20</=200 mg/mL, while 233 (56%) had PC20>/=1600 mg/mL. For those who had a skin prick test, the responders (n=151) and controls (n=151) were found to be matched for age, sex, inhaled corticosteroid dose and number of patients using inhaled corticosteroids. There were significant differences in the number of responders vs. controls in terms of sensitization to house dust mite (77% vs. 62%, P=0.004), aspergillus (19% vs. 9%, P=0.014), cat (61% vs. 48%, P=0.028), total atopic load (493 vs. 380 positive tests, P<0.001) and forced mid-expiratory flow (60% vs. 68% predicted, P<0.001). Sensitization to common aeroallergens increased the likelihood of bronchial inflammation as reflected by bronchial hyper-responsiveness to inhaled AMP, independently of both FEV1 or inhaled corticosteroid use. This in turn suggests an association between allergen exposure and AMP responsiveness in asthmatics. Further prospective long-term evaluation is indicated to assess whether allergen avoidance strategies can modify the airway response.

Pluripotential mesenchymal cells repopulate bone marrow and retain osteogenic properties.

Precursor cells, isolated from bone marrow, can develop into various cell types and may contribute to skeletal growth, remodeling, and repair. The D1 cell line was cloned from a multipotent mouse bone marrow stromal precursor and has osteogenic, chondrogenic, and adipogenic properties. The osteogenic phenotype of these precursor cells is relevant to the process of fracture healing and osteointegration of prosthetic implants. The D1 cells were labeled genetically using a replication incompetent retroviral vector encoding beta-galactosidase, an enzyme which is used as a marker. Labeled cells are readily identifiable by staining with 5-bromo-4-chloro-3-indoyl-beta-D-galactoside and by flow cytometry, and retain the desired osteogenic characteristics in vivo as shown by von Kossa staining, alkaline phosphatase assay, an increase in cyclic adenosine monophosphate in response to parathyroid hormone, osteocalcin messenger ribonucleic acid production, and bone formation in diffusion chambers. In addition, the cells cloned from marrow stroma repopulate the marrow of host mice, persist for several weeks, and retain their osteogenic potential ex vivo. The data suggest that such cells may be used to replenish the number of osteoprogenitors in marrow, which appear to decrease with age, thereby leading to recovery from bone loss and improved bone growth and repair. Labeling these cells creates a model in which to study the potential of such cells to participate in fracture repair, ingrowth around prosthetic implants, treatment of osteoporosis, and to explore the possibility of gene delivery to correct mutations or defects in metabolism that are responsible for certain skeletal abnormalities.

Growth and Function of Isolated Canine Pancreatic Ductal Cells

These studies investigated the growth characteristics and functional properties of isolated canine pancreatic ductal epithelial cells. Cells were isolated from the accessory pancreatic duct and cultured by using three conditions: on vitrogen-coated petri dishes with fibroblast conditioned medium (nonpolarized); in vitrogen-coated Transwells above a fibroblast feeder layer (polarized); or as organotypic rafts above a fibroblast-embedded collagen layer (polarized). Growth characteristics, transepithelial resistances, and carbonic anhydrase and cyclic adenosine monophosphate (AMP) responses were evaluated. Under polarized conditions, the cells grew as monolayers with columnar epithelial characteristics. The monolayers developed high transepithelial resistance and became impervious to the passage of horseradish peroxidase. Epithelial growth factor (EGF) (2 ng/ml) stimulated ductal cell growth and accelerated the formation of a high-resistance monolayer. Forskolin (10 microM) rapidly decreased transepithelial resistance. Carbonic anhydrase activity, which was lower in nonpolarized compared with polarized conditions, was stimulated by carbachol (175 microM). Secretin, however, did not stimulate carbonic anhydrase activity in these cells. Although secretin stimulated adenylyl cyclase activity in early-passage cells, this response was lost in later-passage cells. Both vasoactive intestinal polypeptide (VIP; 1 microM) and forskolin (10 microM) consistently increased adenylyl cyclase activity. Isolated canine pancreatic ductal epithelial cells proliferate in vitro, develop high-resistance epithelial monolayers, and respond to stimuli that activate adenylyl cyclase. These cells should provide a useful model for regulatory studies of ductal cell functions.

3',5'-cyclic adenosine monophosphate-response sequences of the uncoupling protein gene are sequentially recruited during darglitazone-induced brown adipocyte differentiation.

Uncoupling protein-1 (UCP) is uniquely expressed in brown adipose tissue (BAT) and is essential to the thermogenic function of this tissue. The UCP gene is under the control of norepinephrine (NE) via cAMP. However, the precise delineation of the cAMP response sequences and mechanisms whereby cAMP stimulate the gene have remained elusive. A BAT tumor cell line, HIB-1B, can be differentiated into UCP-expressing brown adipocytes. We report here that when these cells are differentiated with a standard differentiation protocol including insulin, T3, hydrocortisone, IBMX, and indomethacin (standard differentiation, StD), cAMP stimulation of the rat UCP gene is largely mediated by an upstream 90-bp sequence -2,399/-2,490 (R90) with a lesser contribution of a downstream sequence -57/+114 (dnCRS). This latter is functional also in non-BAT cells, whereas the cAMP response sequence contained in R90 (upCRS) is BAT-specific. Thiazolidinediones (TZD) are a new group of drugs known to increase sensitivity to insulin and, more recently, to induce adipocyte differentiation (adipogenesis) via PPARgamma. A TZD, darglitazone (darg), can rapidly induce differentiation of HIB-1B cells, as judged by the expression of the adipocyte lipid binding protein (aP2), lipoprotein lipase (LPL), uncoupling protein (UCP) and beta3-adrenergic receptors. UCP messenger RNA (mRNA) responsive to NE is evidenced as early as one day after exposure to darg. While UCP-CAT vectors (+114/-3673 bp of rat UCP gene) are barely responsive to NE in HIB-1B preadipocytes, both darg and StD markedly enhance NE responsiveness of such constructs. However, by 3 days of exposure to darg, the responses were less vigorous than in StD cells (4- to 10-fold vs. 20- to 50-fold), and the deletion of R90 did not affect the response to NE in darg-differentiated cells, whereas this deletion caused a 75% reduction in StD cells. Prolongation of darg exposure to 5-7 days resulted in greater response of UCP mRNA to NE and 50-80% inhibition of the response of UCP-CAT vectors by the deletion of R90. Thus, darg-induced differentiation of HIB-1B cells suggests that the NE-dependent expression of the UCP gene takes place in a step-wise manner: first, the gene is "enabled," as no UCP mRNA is detected in HIB-1B preadipocytes; thereafter and transiently, the response of the gene to NE is sustained by dnCRS; finally, as differentiation progresses, a cell-specific and more powerful cis-acting sequence, upCRS, is recruited, accounting in the fully differentiated cell for most of the response to NE. These results also suggest that TZDs might increase energy expenditure by inducing terminal differentiation of BAT, and that these drugs may be useful in the differential cloning of the factors involved in the recruitment of the BAT specific cAMP response sequence.

Osteoblast precursor cells are found in CD34+ cells from human bone marrow.

It is known that osteoblast precursor cells are found in the low-density mononuclear (LDMN) fraction of human bone marrow (BM) aspirates. The purpose of this study was to investigate whether CD34, a hematopoietic progenitor cell marker, is present on osteoblast progenitor cells. LDMN, CD34+, and CD34- cells were cultured under conditions that promote growth and differentiation of mineral-secreting osteoblasts in a limiting dilution manner. With LDMN cells, osteoblast progenitor cells were found at an average frequency of 1/36,000 cells. With CD34- cells, osteoblast progenitor frequency remained at an average of 1/33,000, similar to LDMN cells. With CD34+ selected cells, osteoblast progenitor frequency increased to an average of 1/5,000. This osteoblast progenitor frequency is maintained in sorted CD34+/CD38+ cells. The osteoblasts generated from CD34+ cells were morphologically normal, and expression of skeletal-specific alkaline phosphatase and osteonectin increased upon differentiation induced by dexamethasone (DEX) treatment. Ultrastructurally, these CD34+ cell-derived osteoblasts displayed osteoblast-specific features. Functionally, these CD34+ cell-derived osteoblasts differentiated with DEX treatment, increased the level of cyclic adenosine monophosphate in response to parathyroid hormone stimulation, increased the level of alkaline phosphatase activity, and increased mineral secretion. These results demonstrate that osteoblast progenitor cells are enriched in the CD34+ cell population from BM and that these progenitor cells can differentiate into functional osteoblasts in culture.

The Autocrine Function of Vasoactive Intestinal Peptide on Human Neuroblastoma Cell Growth and Differentiation

Direct associations between serum concentrations and immunohistochemically detectable vasoactive intestinal peptide (VIP) and maturing neuroblastoma have been documented. Furthermore, VIP has been shown to induce both the growth inhibition and morphological differentiation of cultured human neuroblastoma cell lines. As such, it is hypothesized that VIP may be operative in the autocrine regulation of neuroblastic growth and differentiation. To test this hypothesis, VIP-induced differentiation of human neuroblastoma LA-N-5 cells was performed. Significant concomitant increases in both intracellular and extracellular VIP concentrations were observed. In addition, a marked increase in VIP receptor expression was demonstrated with VIP-induced cellular differentiation. Receptor function was maintained with enhanced expression, as evidenced by an increase in the generation of intracellular cyclic adenosine monophosphate in response to exogenous VIP stimulation. Concomitant enhancement of both intracellular and extracellular VIP expression, coupled with the induction of functional specific VIP receptors during VIP-induced differentiation, provides critical evidence for the autocrine regulation of neuroblastoma maturation by this peptide.

Regulation of corticotropin responsiveness in human fetal adrenal cells

The human fetal adrenal gland exhibits a high rate of steroidogenesis during fetal development and produces the majority of steroids used by the placenta for estrogen synthesis. Corticotropin appears to be the principal hormonal regulator of steroidogenesis in the fetal adrenal gland. However, little is known concerning the regulation of corticotropin receptors. In this study we examined the long-term regulation of corticotropin responsiveness as measured by the ability of human fetal adrenal gland cells to produce cyclic adenosine monophosphate after corticotropin treatment for 3 hours. We also examined the regulation of corticotropin receptors as determined by iodine 125-labeled corticotropin binding to fetal adrenal cells. Fetal adrenal glands were obtained from second-trimester abortuses. The two distinct zones of the fetal adrenal gland, the definitive zone and the fetal zone, were separated and the tissue mechanically dispersed. Freshly isolated cells responded to corticotropin with a sevenfold to tenfold increase in the production of cyclic adenosine monophosphate, indicating a functional corticotropin receptor-adenylate cyclase coupling. However, when either fetal zone or definitive zone cells were grown and passed in monolayer culture, corticotropin stimulation of cyclic adenosine monophosphate production dropped to only twofold. The loss of corticotropin stimulation of cyclic adenosine monophosphate production occurred with a loss of the steroid-metabolizing enzyme 17α-hydroxylase (P-45017α). Because P-45017α expression can be stimulated after treatment of fetal adrenal gland cells with corticotropin or forskolin, we attempted to increase the ability of corticotropin to stimulate cyclic adenosine monophosphate production in a similar manner. After cells were pretreated with corticotropin (0.1 to 100 nmoi/L) or forskolin (0.1 to 100 μmol I L) for 4 days, their ability to produce cyclic adenosine monophosphate in response to corticotropin was examined. Pretreatment with both corticotropin and forskolin caused a dose-dependent increase in the ability of corticotropin to stimulate the production of cyclic adenosine monophosphate. Cells stimulated with corticotropin after pretreatment with forskolin exhibited a 35- to 50-fold increase in cyclic adenosine monophosphate production compared with nontreated cells (≃twofold). Corticotropin pretreatment increased responsiveness to a lesser extent than forskolin pretreatment. The increase in corticotropin responsiveness occurred along with an induction of P-45017α enzyme levels. The effect of pretreatment with corticotropin and forskolin on the binding of iodine 125-labeled corticotropin to definitive zone cells was also investigated. Corticotropin pretreatment increased corticotropin receptor binding 2.8 times; forskolin pretreatment increased corticotropin binding by seven times. These data indicate that corticotropin responsiveness and corticotropin receptors in human fetal adrenal cells are regulated by a cyclic adenosine monophosphate-dependent mechanism. This observation is similar to findings in bovine and ovine adrenal cells where the number of corticotropin receptors is increased by corticotropin or cyclic adenosine monophosphate treatment. (AM J OBSTET GYNECOL 1991;165:1649-54.)

Skeletal contribution of cyclic adenosine monophosphate in response to parathyroid hormone and calcitonin in vivo in the rat.

Cyclic adenosine monophosphate (cAMP) is thought to be a second messenger for the actions of both parathyroid hormone (PTH) and calcitonin (CT). We examined the release of cAMP from rat bone in vivo after administration of synthetic rat PTH-(1-34) (rPTH), synthetic human PTH-(1-34) (hPTH), or synthetic human CT (hCT). Blood from the venous effluent of the femoral bone of rats (bone blood) was drawn at 5 and 10 minutes after the administration of hormones. The cAMP content of the bone blood was then compared to the cAMP content of arterial blood. In both kidney-clamped and non-kidney-clamped rats, hCT led to a significantly greater concentration of cAMP in the bone blood than in the arterial blood. We interpret this to be due to bone production and release of cAMP. Neither hPTH nor rPTH produced a significantly greater amount of cAMP in the bone blood than in arterial blood. These data do not preclude the possibility that there was a production of cAMP within the bone tissue itself after PTH but suggest that there was no release of cAMP from the bone into the bone blood.

Evidence for osteoclast production in mixed bone cell culture.

Osteoclastlike cells (OLCs) were recognized by their morphological, histochemical, and behavioral characteristics. These characteristics were initially observed in freshly isolated osteoclasts when neonatal rat bone cells were introduced into culture. Epithelioid cells proliferated during the first week to form a confluent layer. The cells' alkaline phosphatase content and ability to produce elevated levels of cyclic adenosine monophosphate in response to parathyroid hormone (PTH) led us to tentatively describe them as osteoblastlike cells. When a second addition of neonatal bone cells was made onto this confluent layer, OLCs were produced, their numbers increasing to a maximum 7 days later and dying out 3 weeks later. Mononuclear cells with similar characteristics to these multinuclear OLCs were observed and their production and disappearance closely paralleled that of the OLCs. Fusion of OLCs was observed. The conditions under which OLCs were produced are described.