Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

HIV infection is associated with qualitative and functional immune deficiencies. It has been shown that the in vitro infection of CD4+ cells with HIV was associated with sustained elevation of cAMP and cGMP. In the present report the role of cAMP on HIV replication in MT-4 cells was investigated. The MT-4 cells were infected with HIV (strain 3b), in the presence or absence of agents that increase intracellular levels of cAMP, through different mechanisms. At selected times postinfection, HIV replication was measured by reverse transcriptase activity or HIV P24Ag in culture supernatants. Forskolin (FK, an activator of adenylate cyclase 1-100 microM), Isobutyl-methylxanthine (IBMX, a phosphodiesterase inhibitor, which indirectly increases intracellular levels of cAMP, 30-100 microM) and dibutyryl (db) cAMP (0.1-10 microM) enhanced HIV replication, in a dose-dependent manner. FK, IBMX, and db cAMP enhanced HIV replication by 2- to 10-fold, 4- to 7-fold, and 2- to 6-fold, respectively. Intracellular levels of cAMP were measured by radioimmunoassay and were also enhanced. Since cAMP exerts its catalytic effects through activation of protein kinase (PK) A the effect of H-8 (a specific inhibitor of the cAMP dependent PK A) on HIV replication was simultaneously examined. The H8 at doses of 0.1 to 10 microns inhibited HIV replication by 25 to 99.9%. Moreover H9 inhibited HIV replication in peripheral blood mononuclear cells by more than 90%. The replication of HIV appears to be a cAMP-dependent event, and PK A could possibly be a target for the development of anti-HIV therapies.
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PMID:Human immunodeficiency virus replication: modulation by cellular levels of cAMP. 138

T-cells from human immunodeficiency virus (HIV)-infected patients are characterized by a number of qualitative deficiencies including defective T-cell activation. The latter has previously been shown to be normally regulated by cAMP. In this study the patterns of cAMP and cGMP induction in MT-4 cells following HIV infection were investigated. The MT-4 cells were infected with HIV (strain IIIb) and at selected times postinfection (p.i.), culture supernatants were tested for HIV replication by reverse transcriptase activity or HIV P24 Ag. The cells were also examined for their intracellular levels of cAMP and cGMP by radioimmunoassay. HIV infection was associated with an increase in intracellular levels of cAMP and cGMP. The cAMP was increased 40-fold by Day 8 and cGMP 4-fold by Day 4 Pl. The increase in intracellular levels of the cyclic nucleotides (CN) were virus specific, dependent on virus dosage, genetically conserved among the two fresh patient isolates tested, and were abolished by uv inactivation. An increase in cAMP and cGMP was also observed in other cell lines infected with HIV. The sustained elevation in CN level observed could certainly influence cell activation and HIV replication and may potentially have clinical relevance.
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PMID:Human immunodeficiency virus infection: association with altered intracellular levels of cAMP and cGMP in MT-4 cells. 170 57

Although the biochemical properties of soluble guanylate cyclase (sGC) have been extensively studied, little is known about the regulation of gene expression of sGC subunits by second messengers. cAMP analogues and elevating agents have been previously shown to alter gene expression in vascular cells. The aim of the present study was to investigate the effects of cAMP-elevating agents on sodium nitroprusside-stimulated sGC activity and to correlate activity changes with mRNA and protein levels in cultured rat aortic smooth muscle cells. Pretreatment of cells with 50 to 1000 mumol/L isobutylmethyl-xanthine or 0.01 to 10 mumol/L forskolin led to a time- and concentration-dependent decrease in sodium nitroprusside-induced cGMP accumulation, first evident after 3 hours of pretreatment with forskolin and 6 hours of pretreatment with isobutylmethylxanthine. Incubation of cells with a protein kinase A-selective inhibitor (H89 or KT 5720) partially or fully prevented the downregulation in sodium nitroprusside-induced cGMP accumulation caused by cAMP-elevating agents. Quantification of reverse transcriptase-polymerase chain reaction products by high-performance liquid chromatography revealed that mRNA for both alpha1- and beta1-subunits of sGC were decreased in cells pretreated with isobutylmethylxanthine and forskolin but not with dideoxyforskolin (inactive analogue). Moreover, protein levels for the sGC alpha1 subunit of cells pretreated with isobutylmethylxanthine and forskolin but not with dideoxyforskolin were decreased as indicated by Western blot analysis. These data indicate that cAMP-elevating agents decrease sGC activity, possibly by decreasing mRNA or protein levels or both.
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PMID:Regulation of vascular smooth muscle soluble guanylate cyclase activity, mRNA, and protein levels by cAMP-elevating agents. 755 33

A quantitative reverse transcriptase-polymerase chain reaction for mouse renin mRNA was utilized to study the influence of classic second messenger molecules on renin mRNA levels in primary cultures of juxtaglomerular (JG) cells isolated from the kidneys of C57/B16 mice. We found that forskolin (3 microM), an activator of adenylate cyclase led to proportional increases of renin secretion and renin mRNA levels. The nitric oxide (NO) donor, sodium nitroprusside (100 microM), stimulated both renin secretion and renin gene expression, the effect on secretion being stronger than that on renin mRNA levels. An increase of the extracellular concentration of calcium from 0.5 to 3 mM led to a transient inhibition of renin secretion, followed by a marked stimulation of secretion and to a continuous suppression of renin mRNA levels. These were also decreased by the calcium ionophore A 23187 (1 microM). The membrane permeable 8-bromo-cyclic GMP (100 microM) inhibited basal renin secretion without an effect on renin mRNA levels. The phorbol ester phorbol-12-myristate-13-acetate (1 to 100 nM), which was used to stimulate protein kinase C activity, had no significant effects on renin secretion and renin mRNA levels, neither alone nor in combination with forskolin. These findings suggest that cAMP, NO and calcium are effective regulators of renin gene expression in renal JG cells, in a way that cAMP and NO are stimulators and calcium acts as an inhibitor. Moreover, in these acute experiments there appears to be no obligatory link between the secretion and the expression of renin, suggesting that both parameters are separately regulated.
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PMID:Opposite regulation of renin gene expression by cyclic AMP and calcium in isolated mouse juxtaglomerular cells. 763 56

Atrial natriuretic peptide (ANP) receptors were characterized in rat uterus. The binding of [125I]ANP to uterine membranes was completely competed for by increasing concentrations of unlabeled ANP (Kd = 0.39 nM) and brain natriuretic peptide (Kd = 1.24 nM) and partially by C-type natriuretic peptide (CNP; Kd = 80.4 nM), but not by C-ANF. Also, [125I]Tyr-CNP bound to uterine membranes was completely competed by unlabeled CNP (Kd = 1.12 nM). Cross-linking of [125I]ANP to uterine membranes revealed the presence of one band of 130 kilodaltons, corresponding to the guanylyl cyclase (GC-A and/or GC-B) subtypes of natriuretic peptide receptors. The presence of messenger RNA coding for genes of both GC-A and GC-B receptors was shown by quantitative reverse transcriptase polymerase chain reaction. Furthermore, ANP and, to a lesser degree, CNP stimulated the production of cGMP in rat uterus. Autoradiographic studies localized the highest binding of [125I]ANP in the endometrium, whereas [125I]Tyr-CNP binding was distributed in the endometrium as well as in the myometrium. These results demonstrate that rat uterine ANP receptors are of the guanylyl cyclase-coupled subtypes. The uterus is a target of natriuretic peptides where ANP induces its biological effects through the production of cGMP.
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PMID:Characterization and distribution of natriuretic peptide receptors in the rat uterus. 766 42

The cDNA for a membrane-associated cGMP-dependent protein kinase (cGK II) was cloned from rat intestine using reverse transcriptase PCR and oligonucleotide primers encoding two conserved motifs of known cGMP-dependent protein kinases and subsequently by screening a rat intestine cDNA library. A full-length clone encodes a protein of 761 amino acids with an estimated size of 87 kDa. Sequences of eight peptides from purified pig intestinal mucosa cGK II were found in the derived amino acid sequence of this clone, identifying it as rat intestinal cGK II. Phylogenetic analysis showed that rat intestinal cGK II is less related to mammalian cGK I than to the Drosophila DG1 gene product and most closely related to a recently cloned mouse brain CGKII isoform. Like several other cGK sequences, that of cGK II contained a leucine/isoleucine heptad repeat motif that has been implicated in dimer formation in cGK I. Expression of cGK II cDNA in HEK 293 cells followed by subcellular fractionation revealed cGK II localization in the cell particulate fraction, consistent with the membrane association of endogenous rat cGK II. On Northern blots, the major cGK II poly(A) RNA form was 4.8 kb, with minor forms of 6.2 and 3.1 kb. The cGK II RNA was highly expressed in rat intestinal mucosa and was 20 times less abundant in rat brain and kidney. The localization of endogenous cGK II RNA in rat small intestine was shown by in situ hybridization to be in villous epithelial cells and to some extent in crypt cells.
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PMID:Cloning, expression, and in situ localization of rat intestinal cGMP-dependent protein kinase II. 793 83

Cyclic GMP formation in the rat pinealocyte has generally been thought to involve guanylate cyclases (GC) which are activated via GTP-regulatory proteins following beta 1-adrenergic receptor stimulation. Recent studies have also pointed to a cytosolic GC in these cells whose activity can be elevated by nitric oxide donors. Little attention has been paid to the possibility that pinealocytes might express membrane-bound GC in the form of natriuretic peptide receptors. The present report demonstrates functional membrane GC in rat pinealocytes by (1) cross-linking analyses with radiolabelled atrial natriuretic peptide (ANP); (2) reverse transcriptase polymerase chain reaction (RT-PCR) and DNA blot hybridization with probes for both the GC-A and GC-B forms of the natriuretic receptor; and (3) monolayer cell cultures of pinealocytes, which accumulate cGMP in response to ANP and its related peptides. As the role for cGMP in the rat pineal gland does not appear to be directly coupled to the synthesis of melatonin, the natriuretic peptides may have other regulatory functions in this neuroendocrine tissue.
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PMID:Natriuretic peptides elevate cyclic 3',5'-guanosine monophosphate levels in cultured rat pinealocytes: evidence for guanylate cyclase-linked membrane receptors. 795 2

The mechanism for myometrial quiescence during pregnancy is unknown. cGMP plays an integral role in the relaxation of smooth muscle, and nitric oxide (NO) is the most important endogenous activator of soluble guanylate cyclase. The purpose of this study was to determine the effect of gestational age on myometrial cGMP and NO synthase (NOS) activity in the guinea pig. Myometrial cGMP content (measured by RIA) rose slowly until 0.49 (fraction of pregnancy completed) gestation before abruptly increasing to 200 times the non-pregnant control value. It then declined precipitously after 0.87 gestation. Of the known isoenzymes of NOS, the messenger RNAs coding for both endothelial and neuronal NOS could be amplified from the myometrium of pregnant and nonpregnant animals using reverse transcriptase-polymerase chain reaction, but inducible NOS messenger RNA was not found. Myometrial calcium-dependent NOS activity (measured by the conversion of L-[U-14C]arginine to [U-14C]citrulline) declined slowly with advancing gestation (r2 = 0.096; slope = -0.34; P = 0.01), but never differed significantly from the activity in nonpregnant animals [31.1 +/- 11 (term pregnancy) vs. 56.9 +/- 16 (nonpregnant) pmol/min.g; P = NS]. Calcium-independent activity declined shortly after conception, and then rose toward the nonpregnant level (r2 = 0.19; slope = 0.45; P = 0.0009). However, at no time was it significantly different from that in the nonpregnant animal. Pregnancy had no effect on myometrial L-arginine and L-citrulline content. The administration of L-nitro-arginine methyl ester (200 mg/kg) to inhibit NOS dramatically increased blood pressure and reduced fetal renal NOS activity, but had no effect on the myometrial cGMP content. Estradiol (500 micrograms/kg for 5 days) modestly increased cGMP, but in contrast to many tissues in which estradiol increases NOS, it had no effect on myometrial NOS activity. We conclude that pregnancy dramatically increases cGMP by a mechanism independent of NOS. The stimulus remains to be identified. The temporal change in cGMP concentration is consistent with the hypothesis that cGMP mediates myometrial quiescence during pregnancy.
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PMID:Pregnancy increases guanosine 3',5'-monophosphate in the myometrium independent of nitric oxide synthesis. 798 34

Natriuretic peptides act via receptors with intrinsic guanylate cyclase activity to stimulate cGMP production and are thought to be important regulators of neuroendocrine systems. C-Type natriuretic peptide (CNP) is of particular interest in this regard because the highest tissue concentrations of CNP occur in the anterior pituitary, where it is a highly potent stimulator of cGMP production. Here we show that pituitaries of rats and mice contain abundant CNP prohormone messenger RNA (mRNA), but no atrial natriuretic peptide or B-type natriuretic peptide prohormone mRNAs. Using reverse transcriptase-polymerase chain reaction, both A- and B-type natriuretic peptide receptor (GC-A and GC-B, respectively) transcripts were detected in rat and mouse pituitaries, although only the GC-B mRNA was measurable by Northern blotting. Immunohistochemistry revealed CNP-positive cells in the anterior, but not posterior, pituitaries of rats, and the vast majority of these cells were identified as gonadotropes by colocalization of CNP and LH immunoreactivities. Targeted toxicity using GnRH conjugated to the ricin-A chain was used to test whether gonadotropes are also direct targets for GnRH action. The conjugate dose dependently inhibited the proliferation of alpha T3-1 cells (gonadotrope-derived cells with GnRH receptors), but had no such effect on GH3 cells (which do not have GnRH receptors). Culture of rat pituitary cells with the conjugate caused comparable reductions in CNP-stimulated cGMP production, GnRH-stimulated LH release, and CA2+ ionophore (A23187)-stimulated LH release, but did not measurably alter cAMP production in response to pituitary adenylate cyclase-activating polypeptide. We conclude that CNP is synthesized in the pituitary, where it is located predominantly in gonadotropes, and GC-B receptors expressed in the pituitary mediate the direct effects of CNP in gonadotropes. Together with the recent demonstration of CNP synthesis and action in alpha T3-1 cells, the data suggest CNP to be a novel autocrine regulator of gonadotropes.
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PMID:C-type natriuretic peptide (CNP) in the pituitary: is CNP an autocrine regulator of gonadotropes? 798 73

Our aim was to examine whether the human glomerulus was a target for C-type natriuretic peptide (CNP) and how A, B and C receptors of natriuretic peptides (ANPR-A, ANPR-B, ANPR-C) were distributed in glomerular mesangial and epithelial cells. CNP stimulated cyclic GMP production in cultured human mesangial and epithelial cells with similar threshold concentrations (1 nM) and maximum effects (basal value x 30 at 1 microM). In contrast, atrial natriuretic peptide (ANP) was only stimulatory in epithelial cells. [125I] CNP bound specifically to mesangial cells with a Kd of 0.47 nM and Bmax of 42 fmol/mg. Equilibrium of binding was obtained after four to five hours at +4 degrees C and nonspecific binding represented 10 to 20% of total binding. HS142-1 (100 micrograms/ml), a specific inhibitor of ANPR-A and ANPR-B, suppressed 90% of CNP-dependent cyclic GMP production whereas it had little effect on [125I]-CNP binding, suggesting that C receptors were largely predominant in mesangial cells. No biological effect of CNP on mesangial cells, including change in basal or angiotensin II-induced contractility and inhibition of basal or serum-dependent proliferation, could be demonstrated. Similar results were obtained with 8-bromo-cyclic GMP and sodium nitroprusside. Intraglomerular localization of ANPR-A, ANPR-B and ANPR-C mRNA was studied using reverse transcriptase-polymerase chain reaction with amplification of their corresponding cDNA by different primers. Amplification products were identified on gel electrophoresis by their predicted sizes and sequencing. ANPR-A, ANPR-B and ANPR-C mRNA were present in epithelial cells whereas only ANPR-B and ANPR-C mRNA were detected in mesangial cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of C-type natriuretic peptide receptors in human mesangial cells. 799 93


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